Structural and Biochemical Insights Into Catalytic

Home , Apocarotenal, Apocarotenoid, Lipoxygenase, Retinol isomerase, RPE65, Strigolactone

STRUCTURAL AND BIOCHEMICAL INSIGHTS INTO CATALYTIC

MECHANISMS OF CAROTENOID CLEAVAGE OXYGENASES

XUEWU SUI

Submitted in partial fulfillment of the requirements

For the degree of Doctor of Philosophy

Thesis Advisers: Drs. Philip D. Kiser and Krzysztof Palczewski

Department of Pharmacology

CASE WESTERN RESERVE UNIVERSITY

January 2017 CASE WESTERN RESERVE UNIVERSITY

SCHOOL OF GRADUATE STUDIES

We hereby approve the dissertation of

Xuewu Sui

Candidate for the Doctor of Philosophy degree *.

Philip Kiser (Thesis Advisor)

Krzysztof Palczewski (Thesis Advisor)

Jason Mears, PhD (Committee Chair)

Johannes von Lintig, PhD (Committee Member)

Vivien Yee, PhD (Committee Member)

Matthias Buck, PhD (Committee Member)

Date of Defense

09/02/2016

* We also certify that written approval has been obtained for any proprietary

material contained therein.

I dedicated this work to:

My mentors and friends for inspiration, encouragement and help

My family for love and support

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Table of Contents

List of Tables ...... x

List of Figures ...... xi

ACKNOWLEDGEMENTS ...... xiv

LIST OF ABBREVIATIONS ...... xvi

ABSTRACT ...... 1

CHAPTER 1: INTRODUCTION AND BACKGROUND ON CAROTENOIDS AND

CAROTENOID CLEAVAGE OXYGENASES ...... 3

1.1 Introduction to carotenoids ...... 4

1.2 Introduction to carotenoid cleavage oxygenases ...... 5

1.3 Structural basis for carotenoid cleavage by CCOs...... 6

1.3.1 CCOs display high substrate and cleavage site specificities ...... 6

1.3.2 Conserved 3D architecture of CCOs ...... 9

1.3.3 Structural features in the catalytic center ...... 10

1.3.4 A hydrophobic patch for CCOs membrane penetration...... 13

1.3.5 Substrate binding pocket in CCOs ...... 16

1.4 Proposed CCOs reaction mechanisms ...... 18

1.5 CCOs and human health ...... 20

1.6 Unresolved questions in the CCO field ...... 22

1.7 Goals, experiment outline and rationale for the project ...... 23

Figures...... 26

CHAPTER 2: ANALYSIS OF CAROTENOID ISOMERASE ACTIVITY IN A

PROTOTYPICAL CAROTENOID CLEAVAGE ENZYME, APOCAROTENOID

OXYGENASE ...... 42

2.1 Introduction and background ...... 43

2.2 Experimental procedures...... 46

2.2.1 Protein expression and purification ...... 46

2.2.2 Protein Crystallization...... 47

2.2.3 Enzymatic assay and high-performance liquid chromatography (HPLC)

analysis ...... 48

2.2.4 Purification and mass spectrometric analysis of apocarotenoid product

...... 50

2.2.5 Raman spectroscopy ...... 50

2.2.6 X-ray data collection, structure determination, refinement and analysis

...... 51

2.3 Results ...... 52

2.3.1 ACO purification and enzymatic analysis...... 52

2.3.2 HPLC analysis of the cleavage products ...... 54

2.3.3 In situ analysis of ACO reaction products by Raman spectroscopy ..... 55

2.3.4 Detergents and PEG affect ACO activity ...... 57

2.3.5 Structure of native ACO in the absence of substrate ...... 58

2.3.6 ACO structure in Triton X-100...... 59

2.4 Discussion ...... 61

Tables ...... 66

Figures...... 70

CHAPTER3: KEY RESIDUES FOR CATALYTIC FUNCTION AND METAL

COORDINATION IN APOCAROTENOID CLEAVAGE OXYGENASE (ACO) ..... 88

3.1 Introduction and background ...... 89

3.2 Materials and Methods...... 93

3.2.1 In silico ligand docking ...... 93

3.2.2 Molecular biology, protein expression and purification ...... 93

3.2.3 Enzymatic assays, high performance liquid chromatography (HPLC),

and mass spectrometry (MS) analyses ...... 94

3.2.4 Determination of kinetic parameters ...... 95

3.2.5 Protein crystallization, structural determination, and analysis ...... 96

3.3 Results ...... 97

3.3.1 Identification of potential substrate-interacting residues for mutagenesis

studies...... 97

3.3.2 ACO active site mutants impair catalytic activity to various degrees

without altering regioselectivity ...... 98

3.3.3 Active site mutants primarily affect maximal enzymatic activity rather

than the Michaelis constant ...... 100

3.3.4 The crystal structure of W149A ACO reveals major disruptions in the

substrate-binding cleft and metal coordination...... 101

3.3.5 E150 in the second sphere is critical for metal binding, maintenance of

active site structure and catalytic activity ...... 103

3.4 Discussion ...... 105

Tables ...... 111

Figures...... 117

CHAPTER4: UTILIZATION OF DIOXYGEN BY CAROTENOID CLEAVAGE

OXYGENASES ...... 129

4.1 Introduction and background ...... 130

4.2 Materials and methods...... 133

4.2.1 Phylogeny inference ...... 133

4.2.2 Protein expression and purification ...... 134

4.2.3 Enzymatic assays and rate determination for background oxygen

exchange ...... 135

4.2.4 Isotope labeling study in H218O ...... 137

4.2.5 Sample deoxygenation and isotope labeling study in 18O2 ...... 137

4.2.6 HPLC and mass spectrometric analyses of cleavage products ...... 139

4.2.7 RPE microsome deoxygenation and isomerization activity assays ....139

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4.2.8 Protein crystallization, structural determination and analysis ...... 140

4.3 Results ...... 141

4.3.1 Assessment of apocarotenoid solvent back-exchange...... 141

4.3.2 Apocarotenoid labeling studies in the presence of H218O ...... 142

4.3.3 Apocarotenoid labeling studies in the presence of 18O2 ...... 143

4.3.4 Preparation of highly purified and active NOV2 ...... 143

4.3.5 Assessment of the solvent back-exchange rate for 4-HBA and 3,5-

DHBA ...... 144

4.3.6 NOV2 labeling studies in the presence of H218O ...... 145

4.3.7 NOV2 labeling studies in the presence of 18O2 ...... 146

4.3.8 Activity and structure of T136A-ACO ...... 146

4.3.9 Influence of O2 on RPE65 retinoid isomerase acti vity ...... 149

4.4 Discussion ...... 150

Tables ...... 156

Figures...... 161

CHAPTER5: SUMMARY AND FUTURE DIRECTIONS ...... 180

5.1. Significant of the thesis project ...... 181

5.1.1. Insight into CCOs isomerase hypothesis ...... 181

5.1.2. Structural basis for substrate selectivity and regiospecificity...... 182

5.1.3. Functions of 3-Glu outer Fe-coordination sphere ...... 183

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5.1.4. Resolving the controversy of CCO oxygenation mechanism ...... 184

5.1.5. The requirement of O2 for isomerization reaction by RPE65 ...... 185

5.2. Future directions ...... 186

Bibliography...... 191

List of Tables

Table 2.1. X-ray crystallographic data collection and refinement statistics...... 66

Table 2.2 Experimental and predicted Raman scattering peaks for all-trans-8’- apocarotenol and all-trans-retinal, as well as the di-cis intermediate and 13-cis- retinal product proposed to be generated during ACO-mediated oxidative carotenoid cleavage...... 68

Table 2.3 Inhibition kinetics...... 69

Table 3.1. ACO mutations and their corresponding locations in the active site relative to the docked substrate ligand...... 111

Table 3.2. Steady-state kinetic constants for wild-type and active site-substituted

ACOs ...... 112

Table 3.3. X-ray crystallographic data collection and refinement statistics for ACO mutants ...... 114

Table 4.1. X-ray crystallographic data collection and refinement statistics for

T136A-ACO ...... 156

Table 4.2. Summary of isotope labeling results for ACO...... 158

Table 4.3. Summary of isotope labeling results for NOV2...... 159

Table 4.4. Enzymatic turnover number of ACO and RPE65 before and after deoxygenation treatment...... 160

List of Figures

Figure 1.1. Enzymatic reactions mediated by selected carotenoid cleavage

oxygenases (CCOs)...... 26

Figure 1.2. Structure-based sequence alignment of selected carotenoid cleavage oxygenases ...... 28

Figure 1.3. Crystal structure and topology diagram of cynobacteria ACO, maize

VP14 and bovine RPE65 ...... 30

Figure 1.4. Stereoviews of the catalytic center and electron density around the iron-

binding sites of ACO, VP14 and RPE65...... 32

Figure 1.5. Surface views of the three crystal structures of ACO, VP14 and RPE65

with their hydrophobic patches for putative membrane binding ...... 34

Figure 1.6. Tunnels lead to the active center of ACO, VP14 and RPE65...... 36

Figure 1.7. Monooxygenase and dioxygenase catalytic mechanisms proposed for

carotenoid cleavage enzymes...... 38

Figure 1.8. LCA or RP-associated amino acid substitutions in RPE65 ...... 40

Figure 2.1. CCO substrate binding pockets and proposed isomerase activity .... 70

Figure 2.2. Purification, enzymatic and spectroscopic properties of native ACO 72

Figure 2.3. HPLC analysis of the products generated from ACO-catalyze d

cleavage of all-trans-8’-apocarotenol ...... 74

Figure 2.4. Purification, identification and spectroscopic characterization of 8’-

hydroxy-15’-apocarotenal, the secondary cleavage product of the ACO-catalyze d

reaction ...... 76

Figure 2.5. In situ Raman spectroscopy analysis of ACO reaction products

generated from all-trans-8’-apocarotenol ...... 78

Figure 2.6. Effects of detergents and PEG 3350 on ACO activity...... 80

Figure 2.7. Polyoxyethylene detergent C8E6 non-competitively inhibits ACO catalytic function ...... 82

Figure 2.8. Electron density in the ACO active site ...... 84

Figure 2.9. Structural comparison of ACO crystallized in C8E6 or Triton X-100... 86

Figure 3.1. ACO-catalyzed cleavage reaction and model of the ACO-substrate

complex...... 117

Figure 3.2. Expression, purification and activity of native and mutant ACOs.....119

Figure 3.3. Formation of all-trans-12’-apocarotenal by native and F69A_F303A

ACO ...... 121

Figure 3.4. Steady-state kinetics of native ACO and mutant proteins ...... 123

Figure 3.5. Alterations in the iron center and active site cavity observed in W149A

ACO crystal structures...... 125

Figure 3.6. Disruptions of the active structure and iron coordination center in

E150D and E150Q ACO ...... 127

Figure 4.1. Phylogenic and enzymatic relationships amongst CCOs ...... 161

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Figure 4.2. In vitro isotope-labeling analysis of the ACO-catalyzed reaction .....164

Figure 4.3. HPLC analysis of ACO-catalyzed reaction products and the influence of O2 depletion and supplementation on ACO enzymatic activity ...... 166

Figure 4.4. NOV2 purification and enzymatic characterization...... 168

Figure 4.5. In vitro isotope-labeling analysis of the NOV2-catalyzed reaction ...170

Figure 4.6. HPLC analysis of NOV2-catalyzed reaction products and the influence of O2 depletion and supplementation on NOV2 enzymatic activity ...... 172

Figure 4.7. Active site structure of wild-type and T136A ACO...... 174

Figure 4.8. Activity and in vitro isotope-labeling analysis of T136A-ACO ...... 176

Figure 4.9. Influence of O2 levels on the retinoid isomerase activity of RPE65 .178

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ACKNOWLEDGEMENTS

First I would like to express my deepest gratitude to my thesis advisers, Dr.

Krzysztof Palczewski and Dr. Philip Kiser, for their mentorship and support over the past five years. I am very fortunate to have worked with them. My frequent discussions with Dr. Palczewski enabled me to critically direct my research project.

His suggestions, support and encouragement were essential to the progress in my academic career, and I greatly appreciate his guidance. Dr. Kiser is responsible for guiding me towards studies in crystallography and structural biology, and he taught me the fundamentals of performing rigorous scientific investigations. His deep understanding of protein crystallography and biochemistry inspired me to learn more. After five years of close interactions with him, my appreciation of science and research were greatly enhanced. Beyond science, his philosophy of life also affected me. It has been a great pleasure to work with him, and I cherish the mentorship and friendship he provided.

I am also very fortunate to have worked in Dr. Palczewski’s laboratory. I have benefitted from the professional research atmosphere in the lab. The members in Dr. Palczewski’s lab were always friendly and willing to help me. My project would not have progressed so successfully without the frequent talk, discussions and interactions with members of the laboratory. In particular, I want to thank Drs. Marcin Golczak and Jianye Zhang for their insight and help with my project. I sincerely thank everyone in Dr. Palczewski’s lab for creating such a wonderful research environment and for their help during my PhD training.

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I also wish to thank my committee members, Drs. Jason Mears, Johannes von Lintig, Vivien Yee and Matthias Buck for their guidance throughout my years at Case Western Reserve University. Their constructive comments and feedback regarding my research and education made me a more confident and successful scientist.

A portion of my research were derived through collaborations. Many thanks to Dr. Michael Hendrich and Mr. Andrew Weitz at Carnegie Mellon University and

Drs. Erik Farquar and Wuxian Shi at Brookhaven National Laboratory for the help and effort in our collaborative studies.

Finally, I’d like to use this opportunity to thank my parents and my younger brother. Their love and support always have been a motivating force in my life. No words can express how much I love them. I also want to express my gratitude to my soulmate, Ben Zhang, for her encouragement and partnership in life, and to my parents-in-law for their support and love.

LIST OF ABBREVIATIONS

Å, angstrom;

ABA, Abscisic acid;

ACO, apocarotenoid oxygenase;

atRAL, all-trans-retinal;

atRE, all-trans-retinyl-ester;

ASU, asymmetric unit;

BCO1, β,β-carotene-15,15’-oxygenase;

BCO2, β,β-carotene-9,10 (9’,10’)-oxygenase;

BSA, bovine serum albumin;

BisTris propane, 1,3-bis[tris(hydroxymethyl)methylamino]propane;

C8E4, tetraethylene glycol monooctyl ether;

C8E6, hexaethylene glycol monooctyl ether;

CCD1, carotenoid 9,10(9',10')-cleavage dioxygenase 1;

CCOs, carotenoid cleavage oxygenases;

CMC, critical micelle concentration;

DFT, density function theory;

DMF, dimethylformamide;

HEPES, 2-[4-(2-hydroxyethyl)piperazin-1-yl]ethanesulfonic acid; xvi

HPLC, high performance liquid chromatography;

LB, Luria Bertani;

LC/MS, Liquid chromatography–mass spectrometry;

LCA, Leber congenital amaurosis;

NADH/NAD+, nicotinamide adenine dinucleotides;

NCEDs: 9-cis-epoxy-carotenoid dioxygenases;

NOV2, Novosphingobium aromaticivorans 2;

NinaB, neither inactivation nor afterpotential mutant B;

PDB, protein data bank; PS II, photosystem II;

PEG, polyethylene glycol

RA, retinoic acid;

RAL, retinal;

REs, retinyl esters;

RMSD, root mean square deviation;

ROL, retinol;

RP, retinitis pigmentosa;

RPE, retinal pigment epithelium;

RPE65, retinal pigment epithelium-specific 65 KDa protein;

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SDS-PAGE: sodium dodecyl sulfate polyacrylamide gel electrophoresis;

TCEP, tris-(2-carboxyethyl)phosphine hydrochloride;

VP14, viviparous 14;

XAS, x-ray absorption spectroscopy;

3,5-DHBA, 3,5-dihydroxybenzaldehyde;

4-HBA, 4-hydroxybenzaldehyde;

11cROL, 11-cis-retinol;

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Structural and Biochemical Insights into Catalytic Mechanisms of

Carotenoid Cleavage Oxygenases

ABSTRACT

XUEWU SUI

Carotenoid cleavage oxygenases (CCOs) constitute a large group of evolutionarily conserved enzymes that metabolize a variety of carotenoid and apocarotenoid substrates, including retinoids, stilbenes, and related compounds.

They typically catalyze the cleavage of non-aromatic double bonds by O2 to form aldehyde or ketone products. Their reaction products, denoted as apocarotenoids, serve critical functions in both prokaryotic and eukaryotic cells, including pigmentation, light harvesting, antioxidation, and cell signaling. An RPE65- subgroup of family members expressed in vertebrates catalyze a non-canonical reaction consisting of concerted ester cleavage and trans-cis isomerization of all- trans-retinyl esters, the product of which is essential for visual function. Our understanding of the biological functions of CCOs have progressed significantly in recent years. However, fundamental questions regarding to their catalytic mechanism remain largely unknown.

In this project, we employed Synechocystis ACO that catalyzes canonical cleavage of carotenoids, and Novosphingobium NOV2 which catalyzes the cleavage of stilbene compound, through use of biochemical, structural, and biophysical methods to investigate the conserved catalytic mechanisms. In contrast to findings by others, our biochemical and crystallographic studies of ACO demonstrated that this prototypical CCO member is not an isomerase, as proposed previously. Rather, our results answered the important question of whether isomerase activity is a feature common to all CCOs. Our subsequent structure- directed mutagenesis studies of ACO then provided insights into substrate selectivity and regiospecificity regarding C-C cleavage during catalysis.

Furthermore, our structure-function characterization of mutations of iron- coordination ligands demonstrated the biochemical and structural roles of the conserved 3-Glu, iron-outer sphere in metal coordination among CCOs. Finally, our isotope labeling studies of ACO and NOV2, the latter previously shown to be a monooxygenase, demonstrated that both of the enzymes adopt a dioxygenation mechanism. In conjunction with previous labeling studies of human and plant CCO, our study resolved a long-standing mono- or dioxygenase controversy in the CCO field and revealed a universal dioxygenase reaction mechanism by CCOs. In addition, we also provided clear biochemical evidence showing that the RPE65- mediated isomerization reaction is O2-independent. Collectively, the studies described here provide key insights into the biochemistry of these important metalloenzymes.

CHAPTER 1: INTRODUCTION AND BACKGROUND ON CAROTENOIDS AND

CAROTENOID CLEAVAGE OXYGENASES

This section was previously published in:

Sui, Xuewu, Philip D. Kiser, Johannes von Lintig, and Krzysztof Palczewski.

"Structural basis of carotenoid cleavage: from bacteria to mammals." Archives of biochemistry and biophysics 539, (2013): 203-213.

1.1 Introduction to carotenoids

The most widespread color pigments found in nature, carotenoids comprise a >600 member class of fat-soluble isoprenoid compounds with up to fifteen

conjugated double-bonds. These compounds are synthesized by many types of

organisms ranging from archaea and eubacteria to eukaryotes (algae, fungi and

plants) (for review, see [1]) and perform a host of functions in living organisms that

can be related to the light-absorbing and anti-oxidant properties of their polyene

backbone or their ability to act as signaling molecules. For instance, their well-

known pigment character allows them to impart colors to plants and various

animals (birds, marine organisms, etc.). Such coloring improves the chance of

reproductive success by attracting insects to disperse pollen in the case of plants

or by increasing sexual attractiveness in the case of animals [2]. A second highly

important physiological function pertains to their light capturing, photoprotective

and anti-oxidant properties. Carotenoids are important accessory pigments for

light capture by the light harvesting complex in photosynthetic organisms. The

conjugated double bond system of β,β-carotene can absorb light over a broad

range of wavelengths in blue region of the visible light spectrum and subsequently

transfer that energy to chlorophyll (reviewed in [3]). Their polyene structure also

allows them to react with free radical products and thereby limit damage by

excessive light exposure or free radical metabolites (recently reviewed in [4, 5]).

Carotenoids and their derivatives are also essential for human health playing key

roles in ontogeny, immune function and light perception by the eye. However,

animals (including humans) cannot synthesize carotenoids de novo but instead obtain these compounds from their diet.

1.2 Introduction to carotenoid cleavage oxygenases

In living organisms, carotenoids can be enzymatically converted to a wide array of products. One such group of products, termed apocarotenoids, is generated by cleavage of a double bond within the polyene backbone by molecular oxygen forming an aldehyde or ketone groups at the scissile double bond position.

The first evidence suggesting the existence of a specific carotenoid cleavage enzyme can be traced back to 1965, when two groups reported an enzyme from rat liver and intestine that centrally cleaved β-carotene to form retinal [6, 7].

However, it was not until more than thirty years later, that the first member of this group, named Viviparous 14 (vp14), was cloned and molecularly identified in a screen for viviparous maize seed that showed a decreased level of abscisic acid

(ABA) resulting from the vp14 mutation [8]. This breakthrough facilitated the identification and biochemical characterization of several additional carotenoid cleavage enzymes, not only in plants but also in animals, fungi and bacteria [9-12].

These cleavage enzymes belong to a family of non-heme iron enzymes named carotenoid cleavage oxygenases (CCOs). The overall amino acid sequence identity among family members is variable and can approach random levels; however, the family possesses consensus regions of absolute sequence conservation including the four fully conserved, iron-coordinating His residues.

These enzymes exist in all kingdoms of life except Archaea and play important roles in maintaining carotenoid and retinoid homeostasis. CCOs typically display a

surprisingly high degree of regio- and stereo- specificity for various carotenoid substrates [13]. All conjugated double bonds in carotenoid rigid backbones are potential cleavage sites, and their cleavage by CCOs requires dioxygen resulting in a large variety of apocarotenoids which are involved in various physiological processes.

In recent years, many efforts have been focused on elucidating the CCOs- catalyzed reaction mechanism(s). Recently obtained structural information on

CCOs has provided valuable insights into these processes. Since 2005, crystal structures have been solved for three different CCOs members: apocarotenoid oxygenase (ACO) from cyanobacteria Synechocystis [12], RPE65 from Bos

Taurus [14], and VP14 from Zea mays [15]. These structural data, together with well-documented biochemical and functional properties of these enzymes, provide unprecedented insights into the structural basis for the functional diversity of this protein family.

1.3 Structural basis for carotenoid cleavage by CCOs

1.3.1 CCOs display high substrate and cleavage site specificities

VP14 was the first CCO member found to be involved in ABA synthesis, an important hormone that regulates seed maturation and responses to various stresses in plants [16, 17]. Subsequent study of this recombinant enzyme confirmed that VP14 cleaves 9-cis-violaxanthin at its C11-C12 double bond site to generate xanthoxin, the immediate precursor for ABA biosynthesis [18].

Homology-based analysis with the vp14 sequence helped to identify many other

CCOs in plants. So far two functionally different groups have been documented.

The first group is represented by CCDs (carotenoid cleavage dioxygenases).

Members in this group cleave the polyene backbone either symmetrically or asymmetrically. CCD7 from Arabidopsis cleaves β-carotene asymmetrically at the

9, 10 position, producing β-ionone and apo-10’-β-carotenal; this C27 product can be further cleaved by CCD8 at the 13, 14 position generating the C18 product, 13’- apo-β-carotenal [19]. Recently, CCDs and β-carotene have been shown to play a critical role in the synthesis of the plant hormone strigolactone [20]. Whereas some members recognize and accept specific carotenoids or apocarotenoids, others are more promiscuous in their substrate specificity. For instance, CCD1 from maize specifically cleaves at the 9, 10 position of both cyclic and acyclic carotenoids (e.g. lycopene, β-carotene, and zeaxanthin) [21]. Later research also found the C5-C6 double bond also is a cleavage site for CCD1 [22, 23]. Another group, named

NCEDs (9-cis-epoxy-carotenoid dioxygenases), share a cluster with VP14 and all are implicated in ABA biosynthesis. All NCEDs cleave 9-cis-epoxycarotenoids at the 11, 12 position to yield the ABA precursor xanthoxin [8, 18]. A kinetic study of

VP14 revealed that the 9-cis configuration is strictly required for cleavage activity, but also showed that some flexibility is permitted in the ring structure both distal and adjacent to 9-cis double bond including the presence or absence of than epoxide group [18, 24].

The human genome encodes three CCO members, all of which have been biochemically characterized. Two members, β,β-carotene oxygenase 1 (BCO1) and β,β-carotene oxygenase 2 (BCO2), play critical roles in dietary carotene metabolism, catalyzing the oxidative cleavage of β-carotene at distinct double

bonds in the polyene backbone (Fig 1.1). BCO1 cleaves at the central 15, 15’ site and thus convert β-carotene into two molecules of vitamin A, which is a precursor for visual chromophore (11-cis-retinal) and other signaling molecules such as all- trans-retinoic acid. Therefore, BCO1 plays a critical role in the mammalian visual cycle, embryonic development and regulation of gene transcription, especially in the absence of dietary sources of preformed vitamin A (e.g. retinyl esters). Analysis of substrate specificity revealed that two β-ionone rings of the carotenoid substrate are specifically required for the oxidative cleavage reaction, indicating a limited substrate spectrum for BCO1. By contrast, BCO2 cleaves primarily at the 9, 10 site of a broad range of carotenoid substrates, including β,β-carotene, 5-cis and 13-cis lycopene isomers [25, 26]. Additionally, the enzyme can cleave xanthophylls and

4-oxo-carotenoids [27, 28]. The third member, known as retinal pigment epithelium protein with an apparent molecular mass of 65 kDa (hence the name of RPE65), shares significant sequence homology with BCO1/2 and was identified as an isomerase rather than carotenoid oxygenase [9, 29-31] (Fig 1.2). RPE65 specifically cleaves and isomerizes all-trans-retinyl esters to generate 11-cis- retinol and a fatty acid (Fig 1.1), a key step for regeneration of the visual chromophore, 11-cis-retinal, in vertebrates [29, 30].

Interestingly, CCOs in microorganisms display relatively broad requirements for substrate specificity. One of the best examples is ACO from cyanobacteria, which specifically cleaves apocarotenoids of various polyene chain lengths from C20 to C27 (C4’). This enzyme accepts either terminal aldehydes or alcohol distal to the ionone ring end as well as apocarotenoids with and without a

3-hydroxy group on their β-ionone rings, but selectively cleaves at the 15, 15’

double bond position within these diverse apocarotenoids to generate C20 retinal and a second aldehyde product [12, 13].

1.3.2 Conserved 3D architecture of CCOs

The available crystal structural data reveal a striking architecture consisting

of a rigid seven-bladed β-propeller among CCOs from eubacteria to plants and

mammals (Fig 1.3). In all three solved structures, blades I, II, IV and V consist of

four antiparallel β-strands; blade VI and VII have a single-strand extensions and

therefore contain five antiparallel strands. The third blade of RPE65 contains a

two-strand extension not present in ACO and VP14 structures. This rigid seven β-

propeller scaffold can be viewed as the key structural signature of all CCO family

members (Fig 1.2 and 3).

Although the sequence homology among CCOs are low, their overall 3D

structures are conserved. In all published CCO structures, their secondary

structures start with α-helices. Moreover, α-helixes and various loop regions serve

as transitional elements connecting β-sheets within each blade, thus bringing

separated propeller motifs together (Fig 1.3). The connectivity of the core propeller

fold is identical amongst the three enzymes. Interestingly, although the bottom face

of the propeller has some short α-helixes bridging the strands, most helix elements

are crowded together and their localizations are restricted in the top of the propeller

domain (Fig 1.3). The α-helix elements, together with strands and extended loops,

come together and form a large dome region above the abovementioned seven β-

propeller domain. The catalytic iron is bound on the top face of the propeller on the

propeller axis and is covered by the dome. Unlike the propeller portion, the α-heli x portions in the dome evidence more diversity with respect to both residues and structure (Fig 1.2). The ferrous iron cofactor strictly required for oxidative cleavage is located at the central axis of the propeller. This Fe2+ is coordinated by the Nε

atoms of four absolutely conserved His residues (Fig 1.4). Notably, each innermost

strand from blades II, III, IV and VII in all three structures contributes a single iron-

coordinating His residue. In addition, three of the four His residues hydrogen bond

to a set of three conserved Glu residues that branch from three remaining propeller

blades and form a second coordinating shell with average hydrogen bonding

distances of about ~2.9 Å. Of note, although the side chain of one of the conserved

Glu residues in the VP14 structure (Glu477, PDB accession code 3NPE) points

away from its presumed His hydrogen bonding partner (Fig 1.4B), inspection of

electron density map in this region strongly indicates that the Glu side chain is mis-

modeled in the structure.

1.3.3 Structural features in the catalytic center

The requirement for divalent iron in CCOs is well documented by previous

studies [18, 26, 31-33]. The putative role of Fe2+ is to activate oxygen for cleavage of carotenoid/apocarotenoid substrates [13, 34]. The ferrous iron center in these enzymes is invariably coordinated by four strictly conserved His residues, with three Glu residues forming the second shell (Fig 1.4). The moderate resolution

crystal structures of ACO and RPE65 show average Fe-Nε bond lengths of ~2.1-

2.2 Å, a distance consistent with the 2.15 Fe-Nε bond length measured for RPE65

by X-ray absorption spectroscopy (XAS) [35]. As indicated in the ACO structure,

this platform for iron-binding is rigid and does not change in the iron-free ACO

structure [12]. Mutagenesis studies of the key metal binding first and second

sphere His and Glu residues, respectively, indicate that iron is absolutely required

for CCOs to perform their catalytic roles and that both first and second sphere

ligands contribute to catalytic function and iron binding [36-38].

Several excellent reviews provide detailed discussions about oxygen-

activation by mononuclear non-heme iron containing oxygenases [39, 40]. As in

other ferrous iron containing oxygenase enzymes, the iron cofactor in CCOs is

used to activate triplet oxygen for reaction with singlet organic molecules, a

process otherwise “spin-forbidden” [41]. In all available CCO structures, the iron is

octahedrally coordinated by four His residues. Besides the four His-occupying sites,

two remaining cis-oriented sites that face the active cavity are vacant and available

for exogenous ligands. In the VP14 structure, a 2Fo-Fc density map around the

catalytic Fe2+ center most likely implies a bound water trans to His412 plus a

dioxygen molecule at the two vacant sites (Fig 1.4B). Although a similar dioxygen

or water molecule trans to His183 and His304 is also implied in the ACO structure, it

still remains unclear as to whether a water molecule binds to the sixth coordinating

site of Fe2+ due to the hydrophobic microenvironment resulting from the methyl

group of Thr136 located 4.4 Å away (Fig 1.4A). Similarly, one of the two open Fe2+

coordination sites in the octahedral geometry is partially blocked by the Cγ methyl group of Val134 in RPE65 (Fig 1.4C). A residual 2Fo-Fc signal in the electron density

map with a triangular shape occupies the sixth site in RPE65’s octahedral

geometry. The shape of this density, which is also maintained in its position in

RPE65 structures purified from different detergents [35], indicates a direct interaction between the catalytic iron and the bound retinyl ester-derived free fatty acid product. This RPE65 substrate-interaction model is further supported by an

XAS study, which suggests the presence of a tightly bound carboxylate group at open coordination site(s) of the iron [35].

Often the iron at the active site of mononuclear non-heme oxygenases is coordinated by two or three amino acids, usually a combination of His and other residues (mostly Asp, Glu, and Tyr). The four His coordination system utilized by

CCOs is rare in nature. The imidazole-rich coordination and absence of formally negatively charged direct ligands are likely to greatly stabilize the ferrous form of the enzyme. Indeed, chemical and spectroscopic analyses indicate that CCOs maintain iron in the ferrous form even following aerobic purification in the absence of strong reducing agents. Therefore, what distinguishes CCOs from other iron- requiring proteins is that the former present an unusual iron coordination system.

Thus a coordination in which a protein provides only His residue to coordinate the ferrous ion is rare and observed in only a few cases to date [42-44].

Interestingly, Kloer et al claimed that isomerization activity was embedded in a cyanobacteria ACO because an additional strong electron density appeared at the active center after soaking ACO crystals with the substrate [12]. The crooked rod density could only be fitted by changing the C13-C14 and C17-C18 double bonds from trans configuration to cis. This study provides a novel model for a CCO- mediated carotenoid cleavage reaction that is accompanied by a potential isomerization process. However, both the β-ionone ring and alcohol tail are

invisible and only the central part of the substrate density has been observed. From a chemical perspective, an evident energy source is lacking for the enzyme to overcome the unfavorable energy barrier needed for generation of this double-cis isomer. In addition, the crystals were obtained in the presence of polyethylene glycol and the detergent octylpolyoxyethylene. Those molecules also bear elongated carbon chains resembling the isoprene chain in ACO’s substrate. So the probability that the observed electron density at the active site might simply be a detergent or PEG molecule cannot be rule out. Furthermore, an analysis of ACO- mediated reaction products revealed that only a sparse amount of isomerized products were detected by liquid chromatography [45], which is most likely attributable to thermal isomerization. Therefore, the mechanisms for CCO- substrate interactions and their catalytic processes are open to further clarification.

1.3.4 A hydrophobic patch for CCOs membrane penetration

Unlike other soluble compounds, most substrates for CCOs show high lipophilic features and thus prefer to reside in a water-free environment. Indeed, large amounts of apocarotenoids are found in the thylakoid membrane of plants and microorganisms [46]. And in mammalian cells, carotenoids and their derivatives are stored in lipid droplets called liposomes as well as in other lipid- enriched organelles [47-49]. Thus, these soluble CCO proteins must adopt a mechanism to extract their lipid-soluble substrates. Biochemical studies found that

CCO enzymes display variable levels of aqueous solubility. For example, BCO1 behaves largely like a soluble enzyme [50], whereas RPE65 behaves like an integral monotopic membrane protein [51]. ACO can be expressed in a soluble

form and purified without detergent [12], but it requires detergent for optimal activity

and binds to synthetic liposomes via hydrophobic interactions [52].

Surface analysis of all three CCO structures reveals a similar hydrophobic

nonpolar patch largely consisting of protruding hydrophobic residues (Fig 1.5). The

proposed role of the hydrophobic patch is that it is used by CCOs to dip into the

membrane and extract hydrophobic substrates. In VP14, two antiparallel α-helixe s

mainly consisting of hydrophobic residues (α1-helix from 88 to 108 and α2-heli x

from 222 to 237) form a large putative membrane penetrating patch covering a

surface area of ~2200 Å2 (Fig 1.5B). In addition, a few positively charged residues

(e.g. Arg89) are found in the α1 helix and nearby loop region (e.g. Arg373). Those residues may be involved in the interaction of VP14 with negatively charged lipid in the interior membrane. Unlike VP14, ACO bears a smaller patch with its ~1000

Å2 accessible surface area consisting mostly of Leu and Phe residues, as well as

a few positively charged residues (Lys123 and Arg129, 266) located in the nearby loop

region (Fig 1.5A). Similarly, three groups of residues have been noted in the lipid-

depleted RPE65 structure (residues 196~202, 234~236, and 261~271) where

abundant hydrophobic residues reside including Phe (Phe196, 200, 235, 262, 264), Leu

(Ile 261, 265, 270) and the aromatic residue Trp (Trp268, 271) (Fig 5C). The nonpolar

residue protruding from surrounding loop region (Phe108, 196, 200, 235) may also

contribute to the overall hydrophobicity of ACO and thus mediate the interaction

between the nonpolar patch region and membrane. Also, a number of positively

charged residues are found in those regions. Thus large, nonpolar hydrophobic

patches seem contribute to the overall hydrophobicity of CCO enzymes, explaining

the requirement for detergent for their purification, crystallization and enzymatic

activities. Previous studies suggested that RPE65 is reversibly palmitoylated by

LRAT at Cys231, 329, 330, providing a potential strategy to regulate the membrane

affinity and alter substrate-binding activity of this enzyme [53-55]. However, both

mass spectrometry and mutagenesis studies failed to support a palmitoylation

modification of those Cys residues, and no electron density was found in crystal

structures of RPE65 extracted from its native environment [14, 37, 53, 56].

Therefore, reversible Cys residue palmitoylation is unlikely to regulate RPE65

membrane association and catalytic activity.

Interestingly, a subsequent study revealed a potential role for phospholipids

in modifying RPE65 structure [35]. When the protein was extracted from

microsomes and crystalized in a membrane-like environment, residues involved in

the membrane interaction region adopted a substantially different and more

ordered conformation as indicated by lower active B factors. A distinct change was

the loss of the α5-helix (residues 263~271) accompanied by movement of side

chain Phe264 and Trp268 towards the active center. Several other large movements of side residues including Trp271, Phe200, 196, Lys198 and Asn199 were also observed.

Notably, the completely disordered and invisible chain containing residues

110~126 in the detergent-solubilized RPE65 displayed a weak but continuous

electron density which was orientated parallel and close to the membrane surface.

This segment was previously predicted to anchor RPE65 to the membrane [57], and later studies found that an S-palmitoylated Cys112 in the segment played an important role in mediating membrane association [14, 58]. However, more

biochemical and structural studies are needed to determine whether this native

membrane-induced conformational change also applies to other CCOs enzymes.

1.3.5 Substrate binding pocket in CCOs

Another prominent feature of CCOs is a tunnel extending from outside of

the protein and entering its active center relatively perpendicular to the propeller

axis (Fig 1.6). All tunnels in the three structures pass by the iron metal and end

with an interior Leu residue (Leu446 in VP14, Leu400 in ACO, and Leu439 in RPE65).

This long hydrophobic residue-constituted tunnel acts as a conduit for the passage of lipophilic substrates. The mouths of these tunnels are surrounded by a large hydrophobic patch for membrane insertion mentioned above, thereby providing an ideal lipophilic environment for the substrate. The tunnel hydrophobic residues

(mainly Phe, Val, Leu), together with few of aromatic and hydrophilic residues (Tyr,

Trp and His), work in concert to interact with their hydrophobic carotenoids/apocarotenoids/retinyl ester substrates via van der Waals

(hydrophobic) forces to guarantee both the specificity and correct orientation of substrate for the cleavage or isomerization reactions. Docking experiments modeling 9-cis-violaxanthin into VP14 hydrophobic tunnels revealed that

hydrophobic residues around the methylenecyclohexane group and isoprene

chain interact with substrate via hydrophobic interactions and act also together to

hold the substrate molecule in register for cleavage. Notably, three Phe residues

(Phe171, 411, 589) surrounding the cleavage site and catalytic iron as well as a Val residue (Val478) at the proximal methylenecyclohexane group are key players for

substrate and cleavage site specificity [15]. Mutagenesis studies also

demonstrated the importance of those hydrophobic and aromatic residues in

determining RPE65 isomerase activity, and a recent study indicates that aromatic

residues in the proposed substrate tunnel of RPE65 determine the retinol

isomerization process [59, 60]. Interestingly, most residues determining the shape of hydrophobic conduit are located in the extended loop and α-helical regions, with

only a slight contribution from the well-conserved rigid β-propeller scaffold domain.

Indeed, sequence alignment clearly reveals that regions of the greatest diversity

are within the loop and helix sections (Fig 1.2). The propeller domain in CCOs is

highly conserved among evolutionarily well separated species, whereas the dome

region above the propeller demonstrates the most versatile structural properties.

Therefore, the residues comprising the dome regions appear to be the major

determinants of substrate regio- and stereo-specificity.

Curiously, extra tunnels have been found in all three CCOs structures.

Similarly, the amino residue components of those tunnels are mainly hydrophobic,

with some hydrophilic residues facing the cytosol at the mouth. Three tunnels in

VP14 and two in ACO are connected with their substrate tunnels at the catalytic

center, so these could function as exit conduits for the aldehyde products (Fig 1.6).

Though a long cranked tunnel leads from protein interior to the exterior of RPE65,

several residues separate it from substrate tunnel thereby precluding the passage

of retinoid substrate or product. An improved RPE65 structure revealed well-

ordered water molecules in this tunnel [51]. Therefore, unlike VP14 and ACO, the

RPE65 retinoid substrate entry and product exit most likely occur in the same

tunnel. Of note, one similar conduit along the propeller axis also exists in the ACO

structure. Occluding residues end the conduit shortly before the active metal iron.

The exact function of these tunnels is not clear. Their potential roles may allow

passage to the active center of water or other small molecules (like O2, Fe2+), which

are essential for the enzymatic reaction. But the possibility that such molecules

could also enter the active center via substrate/product tunnels cannot be ruled

out.

Thus when CCOs interact with a membrane, hydrophobic substrates are

extracted and trapped into the nearby substrate tunnel where key residues

promote the accurate orientation of the substrate mainly through hydrophobic

interactions prior to substrate cleavage. In ACO and VP14, reaction products exit

the active center via product exit tunnels, whereas in RPE65 both substrate and

product most likely share the same tunnel to enter and exit the active site.

1.4 Proposed CCOs reaction mechanisms

Despite numerous biochemical studies demonstrating the strict requirement

of ferrous state iron and oxygen for the reaction, the actual oxidative mechanisms

remain controversial. In the monooxygenase reaction, the double bond at the

cleavage site forms an epoxide intermediate with a reactive oxygen species, and

only one of the two oxygen molecules participates in the reaction (Fig 1.7, left

panel). In an in vitro study using purified BCO1 from chicken intestinal mucosa, the

cleavage reaction was initiated in the presence of both 17O2 and H218O.

Subsequent GC-MS analysis of retinol products found almost equal quantities of

oxygen derived from O2 and H2O, providing clear evidence for a monooxygenase

mechanism [50]. Therefore, BCO1 is also named as BCMO1 (β-carotene-15, 15’-

mono-oxygenase) and the latter term is now preferred.

In the dioxygenase reaction mechanism, however, two molecules of oxygen

are involved in attacking the double bond in the substrate and forming a dioxetane

intermediate. This unstable dioxetane species rapidly decays into two aldehyde

products, and water molecules are not required for the reaction (Fig 1.7, right

panel). An isotope labeling experiment involving plant CCD1 from Arabidopsis

thaliana shows that, when performed in an 18O2 atmosphere, 96% of the β-ionone

keto-group and 27% of the aldehyde product were labeled with 18O2 derived

oxygen [61]. Because the aldehyde oxygens in reaction products exchange rapidly

with those of bulk water, this could account for the decreased isotope signal in the

aldehyde product. Therefore, this study favors a dioxygen mechanism. In another

study that took advantage of available CCO structural information, a pure

computational approach suggested that the dioxygenase mechanism is also

preferred for the ACO enzyme [34].

However, due to the nature of oxygen chemical exchange among different

species, isotope-labeling experiments cannot be considered conclusive. For the

monooxygen mechanism [50], there has been criticism about the long incubation

periods used for this reaction. Oxygen exchange between retinal, molecular

oxygen and water during long incubation periods could cause inaccurate

interpretations for equal amounts of formed isolabeled products. Indeed, research

already has shown that most of the oxygen in retinal exchanges with H218O during

14 days [62]. In addition, the alcohol dehydrogenase used to convert retinal to

retinol could potentially scramble the oxygens during the reaction with NADH, and the increased level of NAD+ may exacerbate this process. More data are required to prove that the aldehyde oxygen in retinal does not exchange with the medium during the reaction. And in experiments reportedly supporting the dioxygen mechanism [61], the small fraction of iso-labeled aldehyde product formed constituted weak evidence for the proposed dioxygen mechanism, even though isotopic signals were detected in almost all keto-products. Therefore, while growing body of data have been accumulated in this area, the question of whether

CCOs employ a mono- or dioxygenase mechanism requires further investigation.

1.5 CCOs and human health

So far, three CCO members (BCO1, BCO2 and RPE65) have been identified and characterized in humans. These are key players in regulating carotenoid metabolism and thus are critical for human health. Both BCO1 and

BCO2 act on different double bonds of the β-carotene backbone to generate different products. Vitamin A, generated via central cleavage of β-carotene by

BCO1, is the critical precursor for retinoic acid (RA). RA is known be involved in gene regulation and also participates in a wide range of important physiological processes, including embryonic and fetal development, cell differentiation and metabolic control. Genetic variations of BCO1 caused by mutations or polymorphisms and their effects on carotenoid metabolism in humans have already been described [63, 64]. Although less is known about BCO2, its protective role against carotenoid-caused oxidative stress was implied by a recent study [28].

The physiological functions of BCO1 and BCO2 have been reviewed in detail [65,

66].

RPE65 has gained much more attention because a number of mutations within the RPE65 gene are associated with a severe autosomal recessive early onset retinal dystrophy known as Leber congenital amaurosis (LCA) as well as other milder retinal dystrophies [67, 68]. To date, over 60 different pathogenic mutations have been found in the RPE65 gene spread over all 14 exon as well as intron regions. Although the structure of human RPE65 is currently unavailable, the bovine RPE65 structure provides us with much valuable information about how

RPE65 mutations can affect visual function. Because bovine RPE65 shares 99% sequence identity with human RPE65, it appears highly probable that both enzymes adopt virtually the same chain folds and share the same three dimensional structures. This close relationship enables us to analyze and evaluate mutational effects on a structural level. Because the seven-bladed β-propeller domain is the most conserved portion among CCOs and it serves as the scaffold supporting the active center and the dome of the molecule, any residue substitution causing significant conformational changes in this region should also result in functional defects. Indeed, the mutations of the conserved iron-coordinating His or second coordination sphere Glu residues can produce type 2 LCA or retinitis pigmentosa. Most disease-associated mutations occur within or adjacent to the β- propeller region, especially in blade V and VII, and quite few exist in the helical or loop regions (Fig 1.8). Although no such mutations have yet been found in the membrane binding region of RPE65, some indirect mutations may highlight the

importance of the membrane interaction for proper enzymatic function. For instance, the side chain of Arg91 [69, 70], one of the most frequently affected positions, forms a salt bridge with Glu127, which could be involved in membrane association.

1.6 Unresolved questions in the CCO field

Since the first CCO member VP14 was discovered, more and more members in this family from different species have been identified and characterized, and tremendous progress has been made in defining their roles in carotenoid metabolism and living organisms. Efforts made by structural biologists and breakthroughs in structural studies are vital to further our understanding of

CCOs. Well conserved and specialized structures, together with a unique iron- coordination system are all key properties that distinguish CCOs from other iron- requiring protein families. The hydrophobic tunnels found in all known CCO structures have really improved our comprehension of their substrate specificity, and the hydrophobic patch also explains how CCOs extract their carotenoid/apocarotenoid substrates from at hydrophobic membrane environment.

However, many problems about this enzyme family remain unresolved. For example, although two isotope labeling experiments have been carried out, it is still controversial whether CCOs employ a mono or dioxygen mechanism. Detailed reaction mechanisms and the involvement of oxygen species need to be clarified.

More interestingly, another emerging member named NinaB (denoting neither inactivation nor afterpotential mutant B) is of great current interest. Discovered in insect cells, NinaB combines both cleavage and isomerase activities in single CCO

protein [9, 71, 72]. Therefore, in contrast to the traditional double bond cleavage activity of CCOs, the isomerase activity of RPE65 and isomeroxygenase activity of NinaB separate these specialized members from other canonical CCO family members. One promising way to study catalytic mechanisms by different CCOs is to obtain the crystal structures of definitive enzyme-substrate complexes, but this still remains a formidable experimental challenge because their substrate(s) show a high degree of hydrophobicity and are relatively large in size.

Understanding the substrate specificity and catalytic mechanism of these specialized CCO members also remains a fascinating challenge. Because the roles of RPE65 and BCO1/2 in human health are significant, such studies would definitely help us cope with CCO-related diseases. Moreover, greater efforts are still needed to uncover additional CCOs genes involved in the synthesis and metabolic conversions of both carotenoids and apocarotenoids. And because a growing body of evidence indicates a variety of important biological roles for these hydrophobic isoprenoid compounds, it would not be surprising if nove l psychological functions for carotenoids and apocarotenoids as well as other CCO members, are discovered in the near future.

1.7 Goals, experiment outline and rationale for the project

The primary goal of this project, as stated above, is trying to answer how

CCOs obtain a board substrate specificities while maintain a high degree of regio- and stereo-specificity towards the cleaving site. Ideally, a genuine CCO-substrate complex structure would provide a wealth of insights to those questions. However, although three CCO crystal structures have been solved so far, none of them is

with a bound substrate in the structure. In addition to this, the question of whether

CCOs are using a dioxygenase or monooxygenase mechanism during catalysis and whether the isomerase activity is commonly adopted among CCOs are largely remaining unanswered.

Synechocystis apocarotenoid oxygenase (ACO) is a prototypical CCO that is amenable to biochemical and structure-function studies. When I joined the lab in 2012, the expression vector was ready. So, to realize all above goals, the first task was to set up and optimize the expression and purification protocol for ACO.

Successful in doing this could allow biochemical and structural studies about

CCOs. Finally, I developed an efficient ACO expression and purification protocol with sufficient purified ACO coupled with optimal activity. These progress laid a solid foundation for the entire project.

Though challenging, we also planned to obtain the crystal structure of ACO complexed with apocarotenoid substrate. To increase substrate solubility, we would also collaborate with chemistry department to modify the substrate. To guarantee the success in this complex structure part, we would also perform the biochemical and structural studies on stilbene-cleaving CCOs. Those CCO members catalyzed the carbon-carbon double bond cleavage of stilbene compounds (e.g. resveratrol). The higher water solubility of their substrate could help us to overcome the hydrophobic issues with conventional CCO enzymes that accept carotenoid substrates.

In addition to crystallographic studies, we would also want to determine the mono- or dioxygenase mechanism for CCO-catalyzed reaction, a long-standing

controversy in the field. To overcome the oxygen back-exchange problem, we would use highly purified and active enzyme preparations in our experiments, which would significantly minimize the oxygen-back exchange. We thought those improvements would provide a more clear and definite answer to the questions by performing the isotope-labeling experiments in both H218O-O2 and H2O-18O2 system.

Finally, to test how residues in CCOs’ substrate binding tunnel affect enzymatic properties, we decided to perform structure-directed mutagenesis study on ACO and analyzed the consequences on substrate regioselectivity as well as iron-coordination. Answering those questions would greatly facilitate our understanding in CCOs catalytic mechanisms.

Figures

Figure 1.1. Enzymatic reactions mediated by selected carotenoid cleavage oxygenases (CCOs). Dashed lines in substrates indicate cleavage sites.

Figure 1.1.

Figure 1.2. Structure-based sequence alignment of selected carotenoid cleavage oxygenases. The red background indicates sequence identity, and red letters stand for sequence similarity. All structural elements of VP14, ACO and

RPE65 are shown over the sequence alignment. The strictly conserved iron- coordinating His residues (▲) and their fixating Glu residues (■) are labeled. Dots mark every tenth residue. The alignment was done with T-coffee and the figure was generated with ESPript.

Figure 1.2.

Figure 1.3. Crystal structure and topology diagram of cynobacteria ACO (left), maize VP14 (center) and bovine RPE65 (right). (PDB code: 2BIW, 3NPE and

3FSN). The ferrous catalytic iron is colored in orange. Secondary structural elements consisting of α-helices and β-sheets are colored in blue and green, respectively. The red dashed line in the topology diagram of RPE65 represents the un-modeled loop.

Figure 1.3.

Figure 1.4. Structure of the catalytic center and electron density around the iron-binding sites of ACO (A), VP14 (B) and RPE65 (C). The blue mesh in each structure represents unbiased 2Fo-Fc density maps around the metal iron at a contour level of 1σ. The iron ion is shown as an orange sphere, with the six proposed coordination sites arranged in an octahedral geometry. Four sites are occupied by strictly conserved His residues and the second shell formed by three conserved Glu residues most likely help to stabilize the coordination system. The partially visible electron density in ACO is modeled as the apocarotenol substrate in its cis form. The strong density signals around the iron ion in VP14 were modeled as water (blue sphere) and dioxygen molecules (red stick). Glu477 in VP14 sticks away from its putative coordinating His residue. The residual density in red next to the iron in RPE65 suggests a bound fatty acid molecule.

Figure 1.4.

Figure 1.5. Surface views of the three crystal structures of ACO (A), VP14 (B) and RPE65 (C) with their hydrophobic patches for putative membrane binding. Left, the hydrophobic surface portions of each enzyme are colored in yellow. Right, hydrophobic residues colored in yellow for membrane penetration are shown in each structure.

Figure 1.5.

Figure 1.6. Tunnels lead to the active center of ACO (A), VP14 (B) and RPE65

(C). The red mesh and blue mesh represent the proposed substrate entry tunnels and product exit tunnels, respectively. Residues lining the tunnels are shown as sticks. Hydrophobic residues are colored in yellow, and both charged and polar residues are colored green. The catalytic metal iron is shown as an orange sphere.

Figure 1.6.

Figure 1.7. Monooxygenase and dioxygenase catalytic mechanisms

proposed for carotenoid cleavage enzymes. Except for two vacant sites, the

catalytic metal irons are occupied by imidazole rings from conserved His residues.

A reactive dioxygen binds to the iron and attacks the double bond in the substrate.

In the monooxygenase reaction, an epoxide is formed with involvement of one O2- derived oxygen. Only one oxygen remains in the aldehyde products, with the other entering bulk water. In the dioxyenase reaction, an unstabile dioxetane intermediate is formed, and both oxygen atoms remain in the aldehyde products.

Figure 1.7.

Figure 1.8. LCA or RP-associated amino acid substitutions in RPE65. An

RPE65 topology diagram reveals amino acid positions (colored in red) found substituted in patients with LCA or RP. Numbers indicate positions in the RPE65 amino acid sequence of residues in each secondary structural element. The figure is adopted from Kiser et al [14].

Figure 1.8.

CHAPTER 2: ANALYSIS OF CAROTENOID ISOMERASE ACTIVITY IN A

PROTOTYPICAL CAROTENOID CLEAVAGE ENZYME, APOCAROTENOID

OXYGENASE

This section was previously published in:

Sui, Xuewu, Philip D. Kiser, Tao Che, Paul R. Carey, Marcin Golczak, Wuxian Shi,

Johannes von Lintig, and Krzysztof Palczewski. "Analysis of carotenoid isomerase activity in a prototypical carotenoid cleavage enzyme, apocarotenoid oxygenase

(ACO)." Journal of Biological Chemistry 289, (2014): 12286-12299.

2.1 Introduction and background

Carotenoids are a ~700 member group of diverse, fat-soluble isoprenoid compounds (mostly C40) with up to fifteen conjugated double bonds. The most widespread color pigments in nature, carotenoids exist in all kingdoms of life. The conjugated polyene chain of these compounds endows them with important biochemical properties including visible light-absorption and anti-oxidant activity

[47, 65, 73].

Apocarotenoids are biologically important molecules generated by the oxidative cleavage of carotenoids at specific double bond sites. A group of proteins called carotenoid cleavage oxygenases (CCOs)1 are the main enzymes responsible for catalyzing the cleavage reactions [74]. These enzymes employ a non-heme iron cofactor to activate molecular oxygen for insertion into a carbon- carbon double bond of the carotenoid polyene. The issue of whether these enzymes are mono- or dioxygenases remains contentious and data supporting both cleavage mechanisms exist in the literature for different members of the family

[61, 75]. Carotenoid cleavage activity was first identified in the 1930s [76], but it took another seventy years before the first CCO called VP14 was molecularly identified [8]. Plants express two CCO subgroups: carotenoid cleavage dioxygenases (CCOs) metabolize various (apo)carotenoids, and their products are important pigments, flavor molecules and signaling compounds [20, 77]; 9-cis- epoxycarotenoid dioxygenases (NCEDs) specifically act on 9-cis- epoxycarotenoids to produce xanthoxin, the immediate precursor for abscisic acid biosynthesis [8, 18]. Cyanobacterial CCOs produce retinal chromophore required

for the formation of type-1 holo-opsins [52, 78]. Two human CCOs, BCO1 and

BCO2, are key enzymes involved in dietary carotenoid metabolism. BCO1 symmetrically cleaves β,β-carotene and other pro-retinoid carotenoids to yield all- trans-retinal, which is a key intermediate in the production of visual chromophore in the retina, as well as for generation of the signaling molecule all-trans-retinoi c acid [9, 10, 31, 32, 79]. BCO2 is more promiscuous in its substrate preference and is thought to regulate levels of bodily carotenoids [80, 81], which in excess can be toxic [28, 82].

Interestingly, certain members of the CCO family can also catalyze double bond isomerization concurrently with carotenoid/retinoid cleavage. Such activity was first identified in a retinal pigment epithelium-specific protein named RPE65, which is the retinoid isomerase of the visual cycle [29-31]. This enzyme is unique amongst CCOs in that it catalyzes the non-oxidative cleavage and isomerization of all-trans-retinyl esters to form 11-cis-retinol in an unusual nucleophili c substitution reaction [83]. In biochemical assays RPE65 can also produce 13-cis- retinol [60, 84]. Subsequently, an enzyme that exhibited both carotenoid oxygenase and isomerase activity called neither-inactivation nor after potential B

NinaB was discovered [71, 72]. This enzyme symmetrically cleaves zeaxanthin and simultaneously isomerizes the 11-12 double bond to yield all-trans and 11-cis-

3-hydroxyretinal in an approximate one to one stoichiometry [72]. BCO1 has been shown to partially isomerize 9-cis-β,β-carotene during the oxidative cleavage reaction to yield a less than stoichiometric amount of 9-cis-retinal product [85].

Isomerase activity has also been posited for a cyanobacterial member of the CCO

family called apocarotenoid oxygenase (ACO) [12]. ACO was the first CCO enzyme to have its three dimensional structure determined by X-ray crystallography from C8E4 detergent solution, which revealed a seven-bladed β- propeller architecture with a 4-His-coordinated iron cofactor at its center as the basic CCO fold. In this structural study, a kinked electron density feature was observed in the active site of iron-reconstituted crystals obtained from mother liquor that contained 3-hydroxy-8’-apocarotenol substrate. This density was attributed to bound substrate but a good fit of the apocarotenoid to the density could only be obtained if the compound was converted to a 13,14’-di-cis configuration. Surprisingly, density for the characteristic β-ionone moiety of the molecule was not observed, which made the identity of the compound giving rise to the density uncertain.

To date, three CCO crystal structures are available (Fig. 2.1A) but ACO is the only one for which a purported intact enzyme substrate complex has been structurally described [12, 14, 15]. The bound apocarotenoid model has been used in several studies aimed at elucidating the mechanisms of carotenoid cleavage and carotenoid/retinoid isomerization [34, 59, 86]. As a cyanobacterial enzyme,

ACO is an ancient, archetypical CCO that can be regarded as a primitive scaffold onto which additional or modified structural elements and/or enzymatic activities have been added over the course of evolution. The possibility that (apo)carotenoid isomerase activity was acquired early in the evolution of CCOs is intriguing and has important implications in understanding the mechanism of isomerization by other members of this family such as the visual cycle retinoid isomerase RPE65

(Fig. 2.1B). Therefore, it was of interest to advance our understanding of ACO biochemistry and its catalytic mechanism of apocarotenoid oxidation and isomerization.

Here, we describe a novel expression and purification protocol for ACO that overcomes the difficulties associated with previously described refolding methods and allows recombinant production of native, soluble ACO in E. coli in quantities sufficient for biochemical and high-resolution structural and spectroscopic investigations. Our data highlight the necessity of careful detergent selection in structural studies of CCOs and the binding mode of their substrates.

2.2 Experimental procedures

2.2.1 Protein expression and purification

The bacterial expression plasmid pET3a containing the coding sequence of

ACO (Diox1, GenBank: BAA18428.1) from Synechocystis sp. PCC 6803 was transformed into the T7 express BL21 E. coli strain (New England Biolabs, Ipswich,

MA). Cells were grown at 37 °C to an A600nm of ~0.6 at which time protein expression was induced by the addition of isopropyl β-D-1-thiogalactopyranoside

(Roche, Branchburg, New Jersey) to a final concentration of 10 μM. After 4 h incubation at 28 ºC cells were collected by centrifugation and either flash-frozen and stored at -80 °C or used immediately.

All purification procedures were carried out at 4 ºC. Harvested cells were lysed by three passes through a French press in lysis buffer consisting of 25 mM

4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES)-NaOH, pH 7.0. The lysate was clarified by centrifugation at 186,000g for 30 min. Solid ammonium

sulfate powder (USB, Cleveland, OH) was slowly added within 1 h to the

supernatant with continuous stirring to obtain 20, 30, 40 or 50 % saturated

solutions. Protein precipitation usually occurred within 40 min depending on the

ammonium sulfate concentration. The suspension then was stirred for an

additional 1 h. Ammonium sulfate at 40 % of saturation was judged to produce

optimal ACO precipitation, and this condition was used for subsequent

experiments. The suspension was centrifuged at 46,000g for 20 min, the

supernatant was discarded and the pellet was resuspended in lysis buffer. The

sample was gently rocked for 2 h at 4 °C to allow dissolution of the pellet and then

centrifugated at 186,000g for 30 min to remove any remaining debris. The supernatant was then loaded onto a 120 mL Superdex 200 gel filtration column

(GE Healthcare, Waukesha, WI) equilibrated with a buffer consisting of 25 mM

HEPES-NaOH, pH 7.0, and 1 mM dithiothreitol. Enzymatically active ACO

fractions were pooled, flash frozen and stored at -80 °C for further use.

2.2.2 Protein Crystallization

For crystallization, ACO samples purified by the above method were

concentrated to 20 mg/mL and loaded onto a 25 mL Superdex 200 gel filtration

column (GE Healthcare) equilibrated with a buffer containing 25 mM HEPES-

NaOH, pH 7.0, 1 mM dithiothreitol and 0.8 % (w/v) hexaethylene glycol monooctyl

ether (C8E6). ACO eluted in a single symmetrical peak at ~12 mL. The fractions

were pooled and concentrated to 10 mg/mL. ACO was crystallized by the hanging

drop vapor diffusion method by mixing 1 μL of purified ACO with 1 μL of reservoir

buffer containing 0.1 M 1,3-BisTris propane-HCl, pH 6.0, 22 % - 23 % (w/v) PEG

3350, 0.2 M NH4Cl and 1 mM MnCl2. To crystallize ACO in Triton X-100, we used

0.02 % (w/v) of this detergent instead of C8E6 in the buffer used for gel filtration.

Crystallization conditions for the sample containing Triton X-100 were the same as those for samples prepared in C8E6 Drops were prepared at room temperature and

then incubated at 8 °C. Crystals with a tapered, rod-shaped morphology typically

appeared within 3 - 4 days. Mature crystals were cryoprotected by soaking in the

reservoir solution and flash cooled in liquid nitrogen before X-ray exposure.

2.2.3 Enzymatic assay and high-performance liquid chromatography (HPLC)

analysis

The all-trans-8’-apocarotenol substrate was generated by NaBH4 reduction

of all-trans-8’-apocarotenal (Sigma, St. Louis, MO) in ethanol. The reaction was

stopped by addition of water and the product was extracted with hexane. The

solvent was removed in a SpeedVac and the all-trans-8’-apocarotenol was

redissolved in ethanol. The concentration of product was spectrophotometrically

determined using a molar extinction coefficient of 120,000 M-1cm-1 at 425 nm [87].

Enzymatic activity of the ACO sample was assayed according to a previously published method with some modifications [78]. The enzyme was sensitive to the

Triton X-100 concentration used in the assay with the highest activity at 0.05 %

(w/v), which was the concentration used for enzymatic assays. The presence of detergent would increase the solubility of apocarotenoid molecule and facilitate the delivery of hydrophobic substrate into the active site of ACO. To evaluate inhibition by C 8E6, various C8E6 concentrations were tested in the presence of 0.05 % (w/v)

Triton X-100. Two μL of purified ACO at a concentration of 1 mg/mL was added to

200 μL of reaction buffer consisting of 20 mM 2-[Bis(2-hydroxyethyl)amino]-2-

(hydroxymethyl)-1,3-propanediol-HCl, pH 7.0, and 0.05 % (w/v) Triton X-100. All- trans-8’-apocarotenol in ethanol was then added to initiate the reaction. The reaction mixture was incubated in a Thermomixer (Eppendorf, Hamburg, Germany) at 28 °C with 500 rpm shaking for 3 min and then quenched by addition of 300 μL of methanol. To transform the aldehyde products into corresponding oximes, 100

μL of 2 M hydroxylamine, pH 7.0, was added, and the mixture was incubated for 5 min at room temperature. The products and remaining substrate were extracted in

500 μL of hexane and analyzed by HPLC on a ZORBAX SIL (5 µm, 4.6 x 250 mm) normal phase column (Agilent, Santa Clara, CA). One-hundred μL of hexane- phase extract was injected into the column and eluted isocratically with hexane/ethyl acetate (4:1, v/v) at a flow rate of 1.4 mL/min. The assays were carried out in both ambient light and in a dark room to assess the occurrence of photoisomerization reactions.

For analysis of ACO steady-state kinetics, apocarotenol substrate was added to the reaction tubes to achieve final concentrations ranging from 5 μM up to 200 μM. A burst phase in the kinetics was observed over the first 15 s of the reaction followed by a linear phase up to 2 min for all substrate concentrations tested. Therefore, the reactions were quenched at 15 s, 30 s, 45 s and 1 min for each substrate concentration, and initial velocities were derived by linear regression. Reactions were performed in triplicate for each substrate concentration.

The all-trans-retinal product was converted to the oxime derivative prior to analysis

by HPLC. Analysis of the kinetic data was performed with SigmaPlot (Systat

Software, Inc., San Jose, CA)

2.2.4 Purification and mass spectrometric analysis of apocarotenoid product

The apocarotenoid cleavage reaction was performed with 200 μl of reaction mixture containing 10 mg/mL of ACO. The reaction was quenched after 30 min incubation in the dark, and apocarotenoids were extracted with hexane/diethyl ether in a 1:4 v/v ratio without prior derivatization with hydroxyamine. Solvent was removed by evaporation in a SpeedVac. The sample was redissolved in hexane/ethyl acetate (4:1 v/v) and subjected to HPLC analysis. A hexane/ethyl acetate (4:1, v/v) mobile phase at a flow rate of 1.4 mL/min was used for the separation protocol consisting of: 1) hexane/ethyl acetate (4:1 v/v) for 12 min, 2) a

20 %-100 % linear ethyl acetate gradient developed over 2 min, and 3) 100 % ethyl acetate for 10 min. The peak that eluted at ~17 min was collected and analyzed by LC-MS. The sample was re-injected into a Zorbax Sil (5 µm, 4.6 x 250 mm) normal phase HPLC column (Agilent) and eluted with mixture of hexane/ethyl acetate in a 1:4 v/v ratio. The eluate was directed into an APCI source of an LXQ linear ion trap mass spectrometer (Thermo Scientific, Waltham, MA). Mass spectra were analyzed with the Xcalibur 2.0.7 software package.

2.2.5 Raman spectroscopy

For the Raman spectroscopic study, 100 μL of 2.5 mg/mL purified ACO sample in 25 mM Tris-HCl, pH 8.0, were mixed with an equal volume of buffer containing 25 mM Tris-HCl, pH 8.0, 0.1 % (w/v) Triton X-100 and 50 µM of all- trans-8’-apocarotenol. The reaction was incubated in a Thermomixer at 28 °C with

500 rpm shaking. The reaction was terminated by submerging the tube into liquid nitrogen at different time points ranging from 5 s to 60 min. Experiments were performed in the dark or under ambient light. Frozen samples were lyophilized in the dark overnight, and Raman spectra were recorded from the resulting powders.

Ab initio quantum mechanical calculations of the Raman scattering parameters for substrate and potential products/intermediates were performed on the Case

Western Reserve University (CWRU) high performance computing cluster using

Gaussian 03 (Wallingford, CT) [88].

2.2.6 X-ray data collection, structure determination, refinement and analysis

Diffraction data were collected at the APS ID-24-C and NSLS X29 beamlines. Data for crystals grown in the presence of MnCl2 were collected at wavelengths above and below the iron K-edge to assess the active site iron occupancy, whereas other datasets were obtained at wavelengths where X-ray flux was optimal. Datasets were processed with XDS [89]. The crystals belonged to space group P212121 and were isomorphous to the previously reported ACO crystal structure (PDB accession code: 2BIW) with four monomers in the asymmetric unit [12]. Most crystals examined suffered from epitaxial twinning evident from the presence of two distinct lattices in the diffraction pattern that were rotated 180º about the axis with respect to each other. In many cases the two lattices were sufficiently𝑎𝑎 − 𝑏𝑏 well resolved that their associated intensities could be separately indexed, integrated and then scaled together. Structures were determined either by direct refinement or by molecular replacement using the previously determined Synechocystis ACO structure [12] (PDB accession code:

2BIW) as the starting model in the program Phaser [90]. Initial models were then

subjected to multiple rounds of manual model rebuilding and updating in Coot [91]

followed by restrained refinement in the program Refmac [92]. Refmac input files

were prepared with the CCP4 interface [93]. Bulk solvent parameters were

determined using the “solvent optimize” keyword in Refmac and fixed during

subsequent rounds of refinement. Non-crystallographic symmetry (NCS) restraints

were applied during refinement and gradually loosened or omitted as the model

converged. For the “C8E6 ACO” structure external distance restraints were applied

to the Fe-Nε bonds during refinement to enforce a length of ~2.15 Å. Translation- libration-screw (TLS) refinement of the atomic B-factors (one TLS group per

monomer) was performed near the end of the refinement which further reduced

Rfree [94]. The stereochemical quality of the model was assessed with the

Molprobity server [95]. Data collection and refinement statistics are summarized in

Table 1. Anomalous difference maps were computed using the program ANODE

[96]. Conformational differences between structures were assessed using the

program ESCET [97]. All structural figures were prepared with PyMOL

(Schrödinger).

2.3 Results

2.3.1 ACO purification and enzymatic analysis

The previous structural study of ACO used a protein sample generated by

refolding recombinant protein expressed in E. coli inclusion bodies [12]. ACO has

also been successfully expressed as a soluble GST-fusion protein in E. coli [78].

In both cases, the protein yields were not well documented and multiple purification

steps were required to achieve a homogeneous protein preparation. Thus, we sought to develop a novel, efficient expression and purification method that would generate large quantities of soluble, native enzyme for structural and biophysical studies.

Initially, we observed that native, untagged ACO tends to form large amounts of inclusion bodies when expressed under standard conditions (37 ºC,

0.3 mM IPTG) in E. coli. However, reducing the expression temperature as well as the IPTG concentration during expression dramatically increased the level of soluble ACO without compromising the total protein yield. Interestingly, ACO does not readily bind to common ion exchange or hydrophobic interaction chromatography media under the conditions we tested, prompting us to consider alternative purification methods. We found that ACO could be semi-selectively precipitated from E. coli lysate by ammonium sulfate fractionation. As shown in Fig.

2.2A, ACO precipitates in a stepwise manner with increasing salt concentration whereas many contaminants remain soluble. A 40 % saturated ammonium sulfate solution was found to be optimal for the fractionation. The precipitate from this step was redissolved and then further purified by gel-filtration chromatography. A symmetric peak at ~73 mL that contained apparently pure ACO was collected (Fig.

2.2B). A portion of ACO was aggregated and appeared in the void volume but was not used for further experiments. The typical yield was 5-10 mg of purified protein per liter of E. coli culture.

Our previous XAS studies on this as-isolated sample suggested that the

ACO catalytic iron was in the ferrous state [35]. To confirm the as-isolated sample

obtained by our protocol was enzymatically active, we measured its steady-state kinetic parameters. Values derived from a Michaelis–Menten plot were similar those previously reported for ACO expressed as a GST-fusion protein [78] (Fig.

2.2C). The UV/Vis absorbance spectrum revealed a single peak centered at ~280 nm confirming that no (apo)carotenoids or other chromophores co-purified with

ACO (Fig. 2.2D, upper panel). Additionally, HPLC analysis of a hexane extract of the purified ACO did not reveal any detectable co-purified carotenoids/retinoids as evidenced by the absence of peaks in the 360 and 425 nm chromatograms (Fig.

2.2D, lower panel).

2.3.2 HPLC analysis of the cleavage products

The previously proposed isomerase hypothesis was based on crystallographic data that suggested the presence of a 3-hydroxy-13, 14’-di-cis-8’- apocarotenol intermediate in the ACO active site. However, this proposed activity was not biochemically verified. We reasoned that 13-cis-retinal should be a reaction product if this hypothesis is correct (Fig 2.1B). Indeed, production of trace amounts of 13-cis-retinal was observed in a previous report [78] as well as in our

HPLC analyses as the oxime derivative (Fig. 2.3A, peak a), suggesting potential

ACO isomerase activity. However, the vast majority of retinal product remained in an all-trans configuration (Fig 3A, peak b). This discordant result made us speculate that the trace of 13-cis-retinal oxime could be generated by photoisomerization of the all-trans-retinal product because our experiment was performed under ambient light. To test this, we performed the activity assay and

HPLC analysis under dim light. Consistent with our hypothesis, the 13-cis-retinal

oxime was no longer detected, and all-trans-retinal oxime was the sole cleavage product in these assays (Fig. 2.3A). To exclude any side isomerization reactions that could have occurred during the conversion of aldehyde products into oximes prior to HPLC analysis, which was done to facilitate isomer identification forms, we also directly analyzed the retinal products by HPLC. In this case, the photoisomerization process became even more pronounced, as indicated by the increased amount of 13-cis-retinal (Fig. 2.3B, peak a). Other retinal stereoisomers,

9- and 11-cis-retinal (Fig. 2.3B, peaks between a and b), were also observed.

These isomerized products were not present in control reactions performed in the dark confirming that they indeed arise by photoisomerization. Thus, our data indicated that the generation of 13-cis-retinal during the ACO activity assay was caused by photoisomerization, rather than by the proposed intrinsic isomerase activity.

2.3.3 In situ analysis of ACO reaction products by Raman spectroscopy

13-cis-Retinal, the predicted cleavage product of the putative di-cis- apocarotenoid intermediate, was not detected by our HPLC analyses. However, a previous study suggested that the cis reaction products readily convert back to their all-trans form [12]. This observation could account for the disappearance of the 13-cis-retinal reaction product, prompting us to employ other methods to test the ACO isomerase hypothesis.

Structural differences in the length and geometrical configuration of the carotenoid polyene backbone give rise to characteristic vibrational frequencies that can be spectroscopically detected [98-100]. We employed Raman spectroscopy

to monitor changes in the levels apocarotenoid reactants and products, including geometrical isomers, during the course of the reaction in situ with minimal additional sample workup to avoid any potential back-isomerization.

We measured Raman spectra for pure all-trans-8’-apocarotenol, all-trans and 13-cis-retinal and also for the second apocarotenoid product, 8’-hydroxy-15’- apocarotenal, which was obtained from ACO reaction mixtures and purified by

HPLC (Table 2.2 and Fig. 2.4A,B). Its identity was further confirmed by LC/MS (Fig.

2.4C). Because of difficulties associated with synthesis of the double-cis intermediate, we used the Gaussian package to predict the Raman spectrum of this species ab initio. We also calculated the signals for other molecules that potentially could appear during the reaction. As shown in Table 2, the calculated

Raman peak wavenumbers for each compound were in good agreement with those determined experimentally.

ACO activity assays were performed as described above and terminated at various time points by flash freezing. Following lyophilization, Raman spectra were directly measured on the resulting powders. To prevent any photoisomerization events, the assays were performed in the dark. Also, the 647.1 nm Raman excitation wavelength used in these experiments was far away from the absorbance bands of the polyene and not expected to induce photoisomerization.

The all-trans-8’-apocarotenol substrate exhibited a characteristic peak at 1532 cm-

1 (Fig. 2.5, light grey bar) which was noticeably reduced after a ~10 min incubation, indicating the reaction occurs relatively slowly, a finding consistent with the HPLC data (Fig. 2.2C). The concurrent appearance of product peak at 1580 cm-1 (Fig.

2.5, dark grey bar) corresponding to all-trans-retinal confirmed that the substrate was being productively turned over. Raman peaks for the second apocarotenal product at 1610 cm-1 and 1635 cm-1 started to appear at the same time as those for all-trans-retinal (Fig. 2.5, dark grey bar). Most of the substrate was consumed after a ~60 min incubation. However, during the entire reaction, spectroscopic signals for 13-cis-retinal or the 13,14’-di-cis-8’-apocarotenol intermediate, which would be expected to appear around ~1550-1555 cm-1 were not detected, indicating that no cis products were formed. Collectively, our biochemical and spectroscopic results clearly demonstrate that ACO robustly cleaves its apocarotenoid substrate but does not possess intrinsic isomerase activity.

2.3.4 Detergents and PEG affect ACO activity

Our HPLC and spectroscopy findings did not support the prior hypothesis that ACO possesses intrinsic isomerase activity, which was based on crystallographic data [12]. Because ACO crystallization in this study was carried out in the presence of PEG and the polyoxyethylene detergent tetraethylene glycol monooctyl ether (C8E4) we examined the effects of these compounds on ACO catalytic function. PEG 3350 had little influence on activity up to a concentration of

~10 % w/v (Fig. 2.6A), but at higher concentrations the activity progressively declined, probably due to the ability of PEGs to cause protein aggregation. By contrast, both C8E4 and C8E6 strongly inhibited ACO activity even at concentrations below their critical micelle concentrations (CMC) (Fig. 2.6B, arrows). To examine whether or not this inhibition was a general property of detergents, we measured

ACO activity in the presence of other high-CMC, non-polyoxyethylene detergents.

In contrast to C8E4 and C8E6, CHAPS and CYMAL-4 had much less effect on ACO activity and only partial inhibition occurred at their highest concentrations (Fig.

2.6B). Interestingly, ACO even displayed an elevated activity in the presence of

CHAPS at low concentrations. These data suggest that the linear structure of C8E4 and C8E6 could be responsible for their pronounced inhibitory effects. Indeed, a previously determined structure of iron-free apo-ACO was modeled with a C8E4 molecule bound in its active site, indicating potential competitive inhibition. Next, we studied the mechanism of ACO inhibition by linear polyoxyethylene detergents.

The steady-state kinetic data shown in Fig. 2.7A and Table 3 demonstrated that

C8E6 exerted non-competitive inhibition towards ACO, as indicated by the reduced

Vmax and unchanged Km values. These data suggested that C8E6 might act allosterically to inhibit ACO function although direct binding to the active site could not be excluded, especially at the higher detergent concentrations used for crystallization.

2.3.5 Structure of native ACO in the absence of substrate

In the previously reported ACO structure [12] the proposed enzyme substrate binary complex was generated by crystallizing apo-ACO in the presence of substrate (all-trans-(3R)-3hydroxy-8’-apocarotenol) and detergent (C8E4) and then soaking the resulting crystals in an iron(II)-contai ning solution. Electron density maps computed from X-ray data gathered from these crystals revealed a strong tube-shaped electron density feature in the active site that was attributed to the bound substrate in a di-cis configuration. However, this electron density assignment could not be confirmed because no isomorphous structure obtained in

the absence of substrate was reported. To validate these previous findings we

crystallized native Fe-bound ACO in the presence of the linear polyoxyethylene

detergent C8E6 without the addition of any (apo)carotenoid molecules. C8E6 was

used in the place of C8E4, the detergent used in the original ACO crystallographic study [12], for our studies because it improved crystal reproducibility. Our crystals were isomorphous to the putative ACO-substrate complex crystals and were obtained under similar crystallization conditions. After refinement, the unbiased residual electron density map revealed a tube-like density that filled the substrate entry tunnel and extended past the iron in a curved fashion. Some additional peaks near the iron cofactor, which probably arose from bound water molecules were also observed. The density was highly reminiscent of that reported by Kloer et al.

[12]. To more directly compare the two structures, apocarotenoid molecules in the

previous structure were deleted and residual electron density maps were

calculated. As shown in Fig. 2.8A,B the residual active electron density was

virtually identical between the two structures. The presence of iron in the active

site was confirmed by computing Bijvoet-difference Fourier maps from data

collected above and below the iron K-absorption edge (Fig. 2.8C). These data

strongly indicate that the density previously attributed to isomerized apocarotenoid

substrate arises instead from some other component of the crystallization cocktail.

2.3.6 ACO structure in Triton X-100

The strong inhibitory effects of linear polyoxyethylene detergents on ACO

activity suggested that the bent tube-like density observed in the ACO active site

might represent a bound C8E4 or C8E6 molecule, which could block substrate

binding. Crystallization in the absence of detergent would be a straightforward way to address this possibility, but we were unable to generate crystals from detergent- free mother liquor. Thus, we tested a number of non-linear, ring-containing detergents with bulky hydrophobic moieties including CYMAL-4, CHAPS and

Triton X-100. Of these, only ACO samples containing Triton X-100 yielded diffraction-quality crystals. Despite the change in detergent, these crystals were essentially isomorphous to crystals grown in C8E4 and C8E6 (Table 2.2). Crystals grown in Triton X-100 showed higher resolution diffraction compared to crystals grown in C8E6 with the best crystals diffracting up to 1.9 Å resolution. Additionally, twinning was much less pronounced in these crystals compared to those obtained in C8E6. Electron density maps computed after several rounds of refinement revealed that these crystals also contained residual active site density with a somewhat altered appearance compared to the density observed in the crystals grown in C8E6 (Fig. 2.9A,B). As in the C8E6 crystals, a tube of density was present in the substrate entry tunnel that closely followed the contours of the protein structure. This density merged with a second punctate density feature in direct contact with the iron cofactor, which was best modeled as a coordinated H2O or hydroxide ligand. The tube-like density was somewhat variable in the different protomers of the asymmetric unit. Examination of 2Fo-Fc maps at low contour levels revealed a bent density in the vicinity of the iron somewhat similar in appearance to that of the C8E6 structure residual map density. Notably, this active site density was quite variable from crystal to crystal.

Comparison of these two models by error-scaled difference distance matrix analysis revealed little variability in Cα positions with a few exceptions. One notable difference occurs on the top face of blade II within monomer A, residues

205-212 and 229-234, where the end of the β sheet is shifted away from the substrate entry tunnel in the Triton X-100 versus the C8E6 structures (Fig 9). Some modest ~1 Å shifts in a few active site residue side chains including Phe69 and

Phe113 were also observed between the two structures.

2.4 Discussion

Cis-retinoids have long been known to play important biological roles in all forms of life from cell to cell communication in the case of 9-cis-retinoic acid to photon sensing and signal propagation activities of 11-cis and 13-cis-retinoids bound to visual and non-visual opsin molecules [101, 102]. The elucidation of the

CCO family member, RPE65, as the vertebrate visual cycle isomerase [29, 30, 86] stimulated numerous efforts to understand catalysis by CCOs, in particular the molecular mechanisms of non-photochemical polyene geometrical isomerization

[14, 34-36, 59, 60, 84, 103-105]. Research in this area led to the discovery that

CCOs besides RPE65 also isomerize their substrates during oxidative cleavage

[74, 106]. In some insects, CCOs simultaneously cleave and isomerize all-trans- zeaxanthin to yield all-trans and 11-cis-3-hydroxyretinal, the latter being used to synthesize visual pigments under dark conditions [72, 107, 108]. Cleavage of 9- cis-β,β-carotene by human BCO1 resulted in a lower than expected production of

9-cis-retinal product indicating that this enzyme can isomerize substrate in the cis to trans direction [85, 109]. In the landmark structure determination of

Synechocystis ACO, an active site electron density observed in crystals grown in the presence of apocarotenoid substrate was interpreted as a 13,14’-di-cis- apocarotenoid intermediate, which led the authors to propose the possession of

(apo)carotenoid isomerase as well as oxidative cleavage activity by this enzyme

[12]. The enzyme was therefore expected to produce 13-cis-retinal from all-trans- apocarotenoid substrates, but this supposition was not biochemically evaluated

[13]. Moreover, the ambiguous appearance of the density has led investigators to question its identity [74, 77]. From a biological point of view, the proposed isomerase activity of this enzyme is unexpected given that Synechocystis opsins, which utilize retinal generated by ACO, are converted to ground-state holo- pigments by the binding of all-trans rather than 13-cis-retinal [102]. Given the extensive use of the ACO-substrate model in studies aimed at understanding CCO substrate specificity and catalytic mechanism, we considered the validation of this structure and the biochemical predictions based upon it to be an important task.

To facilitate the study of this enzyme we first developed a novel expression and purification protocol. In contrast to previous methods that relied on a fusion protein strategy [78] or refolding from E. coli inclusion bodies [12], we were able to express native, untagged ACO in a soluble form in E. coli and purify it to homogeneity by a simple two-step procedure involving ammonium sulfate fractionation and gel filtration chromatography. Through this procedure we could obtain quantities of pure ACO sufficient for future high resolution biophysical studies, which are in general lacking for this class of enzyme. The preparation was enzymatically active and the metal center was fully occupied by iron [35]. We

observed that freshly purified ACO sample displays 2.6 - fold higher activity but 2.7

- fold decreased Km for its apocarotenoid substrate as compared with an aged, air- exposed sample (compare Figs. 2C (aged for 2 weeks at 4 ºC) and 7A (fresh)).

Ferrous iron is known to be required for the activity of CCOs [18, 33, 86]. Because

ACO was purified under aerobic conditions in the absence of reducing agent, we

expect that the diminshed activity could be the result of iron oxidation.

Nevertheless, the ACO iron(II) center is remarkably stable compared to many other

non-heme iron(II)-dependent enzymes that often must be purified anaerobically to

prevent iron oxidation [110, 111]. Analysis of ACO reaction products generated

from all-trans-apocarotenoid substrate both by standard HPLC as well as by in situ

Raman spectroscopy revealed exclusive production of all-trans-retinal. We found

that the previously reported traces of 13-cis-retinal produced during the assay

could be attributed to photoisomerization of all-trans-retinal under normal lighting

conditions [12, 78]. Surprisingly, we also found that the polyoxyethylene detergent,

C8E4, used in the original structural study strongly inhibits ACO activity indicating that the crystallized protein might be catalytically inactive. Indeed, using Raman spectroscopy we could not detect in crystallo production of all-trans-retinal from

ACO crystallized in the presence of C8E4. Inhibition was observed for a related

linear polyoxyethylene detergent, C8E6, but was absent or much less pronounced for other bulky detergents depending on their concentration.

To directly examine the possibility that the active site electron density in the previously reported structure represents bound apocarotenoid substrate or an isomerized intermediate, we crystallized native ACO under conditions similar to

those previously reported except that apocarotenoids were not present during protein purification or crystallization. The active site electron density observed in these crystals was strikingly similar in appearance to that reported by Kloer et. al. despite the absence of added carotenoids demonstrating that the density was not attributable to bound substrate (Fig. 2.9A) [12]. We have observed that this density does not change in appearance when our ACO crystals are soaked with all-trans-

8’-apocarotenol. A second structure obtained with Triton X-100, a detergent that supports ACO activity, substituted for C8E6 also displays residual active site density albeit with a slightly altered appearance (Fig. 2.9B). Given the similarity in the density between the two structures, we feel that it is unlikely to represent bound detergent. Instead, this density might arise from another component of the crystallization cocktail, possibly a contaminant bound in the active site. It could also simply represent a chain of partially ordered, hydrogen bonded water molecules.

The relatively narrow dimensions and hydrophobic nature of the substrate entry tunnel might cause an ordering of water molecules that could occupy the cavity to prevent formation of an energetically unfavorable vacuum [112, 113].

The structure reported by Kloer et al. is the only CCO for which an experimental enzyme-substrate was proposed [12]. In light of the data presented here the model of the bound substrate can only be considered an educated guess that is wrong in detail (i.e. the configuration of the substrate is incorrect). Thus, caution should be applied when using this model for developing hypotheses of

CCO substrate specificity and catalytic function. The kinetics data presented in this study emphasize that proper selection of detergent will be critical for obtaining a

genuine CCO-substrate complex. CCOs possess a conserved hydrophobic patch on their surfaces near their substrate entry tunnels that facilitates extraction of lipophilic substrates from membranes [74, 83]. Inclusion of detergent seems to be critical for the crystallization of ACO and RPE65, probably due to its ability to bind the hydrophobic patch and prevent non-specific protein aggregation. Interestingly, the structure of the 9-cis-epoxycarotenoid dioxygenase, VP14, was determined in the absence of detergent [15]. In this structure hydrophobic surfaces from symmetry-related molecules pack together and shield each other from the solvent.

Despite inclusion of carotenoids in the crystallization solutions such compounds were not identified in the active site of this enzyme [15]. In this case, delivery of highly hydrophobic carotenoids to the enzyme may have been impeded by the absence of detergent micelles.

In summary, using this highly-purified, enzymatically active preparation we investigated the enzymatic activity of ACO by HPLC and Raman spectroscopy.

Both methods revealed robust production of all-trans-retinal from all-trans-apo-8’- carotenol by ACO. Traces of cis isomers of the product were observed only in reactions performed under ambient light suggesting partial photoisomerization of the retinal product. We observed a pronounced inhibitory effect of linear polyoxyethylene detergents used for ACO crystallographic studies on all-trans- retinal production by ACO. We showed that the ACO active site density previously attributed to an isomerized apocarotenoid intermediate originates from non- substrate molecule(s).Thus, obtaining an intact CCO-substrate complex remains a challenge for future studies.

Tables

Table 2.1. X-ray crystallographic data collection and refinement statistics.

Data collectiona

Data set name C8E6 crystal Tri ton X -100 crystal

Beamline X29 NECAT 24-ID-C

Wavelength (Å) 1.7308 0.9793

Space group P212121 P212121

a = 118.56 a = 118.47 Unit cell parameters (Å) b = 125.12 b = 125.39 c = 203.01 c = 202.54

Resolution (Å) 50-2.8 (2.97-2.8)a 50-2.0 (2.05-2)

Unique reflections 75089 (11789) 200469 (14869)

Completeness (%) 99.7 (98.2) 98.7 (99.8)

Multiplicity 11.7 (9.9) 7.3 (5.9)

10.1 (1.4) 11.74 (1.56)

RmergeI (%)b 27.2 (168.5) 8.6 (97.7)

RmeasI (%)b 28.5 (177.7) 9.3 (107.7)

CC1/2 (%)b 99 (54.2) 99.8 (54.5)

Wilson B-factor (Å2) 45.9 46.4

Refinement

Resolution (Å) 48.6-2.8 48.6-2.0

No observations 71479 190607

Rw ork/Rfree (%)c 21.1/23.6 17.7/20.5

No atoms

Protein 15072 15154

Water 87 875

Metal/ion 4 Fe, 3 Cl 4 Fe, 2 Cl

B-factors (Å2)

Protein 51 47

Water 35 47

Metal/ion 30 (Fe), 55 (Cl) 34 (Fe), 48 (Cl)

RMS deviations

Bond lengths (Å) 0.011 0.011

Bond angles (°) 1.41 1.44

Ramachandran plotd

Favored/outliers (%) 97.5/0 98/0

PDB accession code a Values in parentheses are those for the highest resolution shell of data b As calculated in XDS c As calculated in REFMAC d As defined in MolProbity

Table 2.2 Experimental and predicted Raman scattering peaks for all-trans-

8’-apocarotenol and all-trans-retinal, as well as the di-cis intermediate and

13-cis-retinal product proposed to be generated during ACO-mediated oxidative carotenoid cleavage.

Experimental peak Calculated peak Compound wavenumber (cm-1) wavenumber (cm-1) a all-trans-8’-apocarotenol b 1532 1526

13,14’-di-cis-8’-apocarotenol ND c 1552

all-trans-retinal 1580 1581

13-cis-retinal 1555 1550

8’-hydroxy-15’-apocarotenal 1599 1619 d a As calculated by Gaussian 03 [88] b See Figure 2.1 for chemical structures c Not determined d Calculated signals for this all-trans-apocarotenoid product and its cis- isomer are indistinguishable

Table 2.3 Inhibition kinetics.

Km (app) (µM) Vmax (app) (pmol/sec) Control 121.03 ± 24.13 21.02 ± 1.95

0.1 CMC C8E6 126.79 ± 21.49 16.21 ± 1.31

Figures

Figure 2.1. CCO substrate binding pockets and proposed isomerase activity.

A. Clipped views of the substrate binding clefts of Synechocystis ACO, bovine

RPE65 and maize VP14. Arrows indicate substrate entry sites for each CCO. The iron centers are shown as orange spheres. B. RPE65, the all-trans to 11-cis- retinoid isomerase of the vertebrate visual cycle, is an atypical member of the CCO family that catalyzes a coupled ester cleavage/isomerization reaction rather than oxidative cleavage. NinaB, the sole CCO expressed in insects, cleaves and isomerizes zeaxanthin and related carotenoids to generate all-trans and 11-cis- retinoids in a ~1 to 1 ratio. ACO, a prototypical CCO expressed in cyanobacteria, was suggested to possess both oxidative cleavage and carotenoid isomerase activity on the basis of the appearance of electron density in the active site of substrate-soaked crystals. Whereas carotenoid cleavage activity of this enzyme is well-established, its ability of the enzyme to isomerize carotenoids has not been biochemically verified. Red wavy lines indicate the location of the scissile bond for each cleavage reaction. Dashed double bonds in each substrate indicate known or potential sites of geometric isomerization. Percentages indicate yields for each product.

Figure 2.1.

Figure 2.2. Purification, enzymatic and spectroscopic properties of native

ACO. A. SDS-PAGE analysis of fractions precipitated by ammonium sulfate at different percent saturation levels. Proteins were separated on a 4 %-20 % 2-

[Bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)-1,3-propanediol /glycine gradient gel and were visualized by Coomassie R-250 staining. The arrow indicates the position, at about 54 kDa, where ACO migrates. B. The 40 % saturation precipitate shown in panel A was redissolved and further purified by gel filtration chromatography. Protein eluting within the peak indicated by an arrow was pooled, concentrated and used for experiments. The purity of the final sample was evaluated by SDS-PAGE followed by Coomassie R-250 staining (inset). C. Initial velocity vs. all-trans-8’-apocarotenol concentration curve for the purified ACO sample showing typical Michaelis-Menton kinetics. Km and kcat values derived from the curve are shown in the inset. D. (Top) The UV-Vis absorbance spectrum of a purified ACO sample at a concentration of 1 mg/ml (upper panel) was recorded immediately after the purification. (Bottom) HPLC analyses of the purified protein sample. Hexane was used to extract any possible apo/carotenoids from 200 μl of purified ACO at 10 mg/mL. Traces a and b were monitored at 360 nm and traces c and d were monitored at 425 nm. Negative control samples in which protein sample was omitted from the extraction procedure are shown in traces b and d.

No apo/carotenoids were detectable at the wavelengths tested in this assay.

Figure 2.2.

Figure 2.3. HPLC analysis of the products generated from ACO-catalyzed cleavage of all-trans-8’-apocarotenol. A. The aldehyde products generated by

ACO were converted into oxime derivatives by treatment with hydroxylamine prior to HPLC analysis. Exclusive production of all-trans-retinal was observed when the assay and HPLC analysis were performed under low illumination conditions. A small peak with a retention time of 8.7 min corresponding to syn-13-cis-retinal oxime was observed when the assay and analysis were conducted under ambient lighting conditions indicating photoisomerization of a small portion of all-trans- retinal product. Spectra for each peak are shown in the panels on the right. The analysis shown in panel B was conducted exactly as in A but reaction products were analyzed in their unaltered aldehyde forms. The presence of larger amounts of retinal geometric isomers in the sample generated under ambient lighting conditions indicates that photoisomerization occurs more readily in the retinal products as compared to the oxime derivatives. Note that peaks between a and b correspond to 11- and 9-cis-retinal, respectively. Peak a: syn-13-cis-retinal oxime

(in A) or 13-cis-retinal (in B); Peak b: syn-all-trans-retinal oxime (in A) or all-trans- retinal (in B); Peak c: all-trans-8’-apocarotenol.

Figure 2.3.

Figure 2.4. Purification, identification and spectroscopic characterization of

8’-hydroxy-15’-apocarotenal, the secondary cleavage product of the ACO- catalyzed reaction. A A 200 μl ACO sample at 5 mg/ml was incubated with 200

μM all-trans-8’-apocarotenol for 45 min in the dark. Apocarotenoids then were extracted and analyzed by HPLC. Peaks a and b eluted at 4.2 min and 8.8 min and corresponded to all-trans-retinal product and substrate, respectively. Peak c eluted in 100 % ethyl acetate at 17.3 min and corresponded to 8’-hydroxy-15’- apocarotenal on the basis of its absorbance spectrum (inset). This peak was collected for further analysis. B. A typical Raman spectrum of the 8’-hydroxy-15’- apocarotenal product. The collected peak c fraction was dried in a SpeedVac, and the resulting powder was dissolved in water prior to the Raman assay. The compound displayed a characteristic spectrum with prominent peaks at 1599 cm-1 and 1635 cm-1. C. MS analysis of peak c revealed a major peak at m/z 167.2 corresponding to the protonated form of 8'-hydroxy-15'-apocarotenal. Tandem MS analysis of the 167.17 peak showed a major fragmentation product at an m/z of

149.17 representing loss of water from the parent ion.

Figure 2.4.

Figure 2.5. In situ Raman spectroscopy analysis of ACO reaction products generated from all-trans-8’-apocarotenol. Reactions were quenched at 5 s, 10 min, 30 min and 60 min by emersion in liquid nitrogen. Samples then were lyophilized and Raman difference spectra were measured. All-trans-8’- apocarotenol and all-trans-retinal gave rise to prominent peaks at 1532 cm-1 (light grey bar) and 1580 cm-1 (right dark grey bar), respectively. Peaks arising from the secondary product at 1610 cm-1 and 1635 cm-1 (left dark grey bar) become detectable at 30 min. The reaction was nearly complete after the 60 min incubation.

Notably, the spectra did not exhibit peaks at 1552 cm-1 (calculated) and 1555 cm-

1 where the hypothesized 13,14-13’,14’-di-cis-apocarotenoid intermediate and 13- cis-retinal products were expected to appear.

Figure 2.5.

Figure 2.6. Effects of detergents and PEG 3350 on ACO activity. A. Inhibitory effects on ACO exerted by PEG 3350, the precipitant used for the growth of ACO crystals, occur at relatively high concentrations. B. The activity of ACO in 0.05 %

(w/v) Triton X-100, the detergent normally used in ACO activity assays, was severely inhibited by addition of the linear detergents C8E6 and C8E4 used for crystallographic studies of this enzyme, even at concentrations below their CMC values. Conversely, bulkier non-linear detergents such as CYMAL-4 and CHAPS had a much less pronounced effect on ACO activity. Error bars represent standard deviations computed from three independent experiments.

Figure 2.6.

Figure 2.7. Polyoxyethylene detergent C8E6 non-competitively inhibits ACO

catalytic function. A. A Michaelis-Menten kinetic plot of ACO in the presence of

varying C8E6 concentrations. The decreased Vmax and unchanged Km (shown in

Table 3) at a concentration of C8E6 equal to 0.1 times its CMC suggested a non- competitive mode of inhibition. Note that the reaction displayed more complex kinetics and the velocity didn’t plateau in the presence of C8E6 at a concentration

of 0.2 times its CMC, which precluded derivation of Vmax and Km values from these

data. B. C8E6 dose-dependently inhibited ACO activity. The IC50 for C8E6 was 2.15 mM, which corresponds to ~0.22 times its CMC value.

Figure 2.7.

Figure 2.8. Electron density in the ACO active site. A. The 13,14’-di-cis-3- hydroxy-8’-apocarotenol molecules and water molecules in close proximity to the catalytic iron were removed from the deposited ACO structural model (PDB accession code: 2BIW), and residual (mFo-DFc) electron density maps were calculated in REFMAC. The density was averaged over the four ACO monomers in the asymmetric unit using Coot. Density within 3 Å of the proposed substrate position is shown in green at a contour level of 5σ. Residues within 5 Å of the density are shown as sticks. The ferrous iron is shown as an orange sphere. B. A strikingly similar unbiased density feature is present in crystals grown in the absence of substrate. Representations are the same as in panel A. C. Bijvoet- difference Fourier maps were calculated from data collected above (blue mesh) and below (green mesh) the iron K absorption edge, each contoured at the 6 σ level. The large difference in anomalous scattering confirms the presence of iron in the active site of “as-isolated” ACO.

Figure 2.8.

Figure 2.9. Structural comparison of ACO crystallized in C8E6 or Triton X-100.

Shown are unbiased mFo-DFc (green mesh) and 2Fo-Fc (blue mesh) electron

density maps contoured at 3σ and 1σ, respectively, displayed within the active site

regions of ACO crystallized in the presence of A C8E6 or B Triton X-100. The

densities are similar in appearance with the exception of a break in the density

after it passes the iron center in the Triton X-100 structure. Black arrows indicate

residual electron density that likely represents an iron(II)-bound water or hydroxide

ligand. C. Difference distance matrix analysis of the C8E6 and Triton X-100 ACO

structures. The triangular matrix below the diagonal is an error-scaled difference

distance matrices generated by pair-wise comparisons of NCS-related monomers

using the program ESCET, whereas the upper matrix is an absolute difference

distance matrix. Red and blue elements stand for structural expansion or

contraction, respectively. The intensity of the color is proportional to the changes

in distance, and grey elements indicate structural invariance. Secondary structural

elements are shown beneath the matrix: open boxes are helices, black boxes are

β-sheets. D. Structural superposition of the C8E6 (yellow) and Triton X-100 (blue)

showing a rigid body movement of the top face of propeller blade II in chain A of

the structure. Consequently, the mouth of the protein is more in the Triton X-100

structure compared the C8E6 structure in this particular monomer. Small changes were also evident in regions of the protein predicted to contact membranes (blue arrows). The catalytic iron is shown as an orange sphere. The green arrow delineates the substrate entry tunnel that leads to the catalytic center.

Figure 2.9.

CHAPTER 3: KEY RESIDUES FOR CATALYTIC FUNCTION AND METAL

COORDINATION IN APOCAROTENOID CLEAVAGE OXYGENASE (ACO)

This section was submitted to the Journal of Biological Chemistry at the time of writing this thesis

3.1 Introduction and background

In metazoans, vitamin A (all-trans-retinol, ROL) and its metabolites, collectively referred to as retinoids, participate in many essential physiological processes, including embryonic development, growth, immune function, reproduction and vision [114-117]. Therefore, a sustainable supply of vitamin A is critical to fulfill the diverse biological functions associated with retinoids. However, the lack of a de novo vitamin A biosynthetic pathway in animals necessitates their dietary intake of provitamin A carotenoids or retinyl esters (REs) as vitamin A precursors [118]. In mammals, β-carotene, the most abundant pro-vitamin A carotenoid in nature, is absorbed by intestinal mucosal cells and oxidatively metabolized into vitamin A-aldehyde (all-trans-retinal, RAL), which can be successively reduced to vitamin A and stored in the liver as fatty acid retinyl esters

(e.g., retinyl palmitate) [119-121]. Oxidative cleavage of the carotenoid polyene chain is catalyzed by a family of non-heme iron-dependent enzymes known as carotenoid cleavage oxygenases (CCOs) that are found in all kingdoms of life [13,

74, 77]. Besides their involvement in retinal formation, CCOs generate several other important apocarotenoids including abscisic acid, strigolactones, pigments and volatiles in plants and degrade stilbenoid compounds such as resveratrol and it derivatives as well as lignin catabolites in bacteria and fungi [77, 122-125].

Although CCOs can often cleave multiple substrates, they generally display high regio- and stereo-selectivity with respect to the cleavage site within the carotenoid polyene chain as well as the polyene isomeric configuration [13, 77].

For example, human BCO1, a retinal-forming CCO, catalyzes symmetrical

cleavage of different all-trans cyclic carotenoids at the central double bond position

(C15-C15’ according to traditional carotenoid carbon numbering) of the polyene chain [6, 7], whereas BCO2, another human carotenoid metabolizing CCO, is capable of cleaving not only cyclic but also acyclic carotenoids asymmetrically (C9-

C10 position) [13, 26, 126]. Not limited to mammals, these distinct enzymatic features (i.e., substrate promiscuity coupled with regio- and stereo-selectivity) are also commonly found in plant and bacteria CCOs [45, 52, 77]. For example, a plant

CCO called VP14 that plays a critical role in abscisic acid biosynthesis cleaves 9- cis-epoxycarotenoids selectively at the C11-C12 polyene double bond [18, 24].

Interestingly, certain CCOs exhibit isomerase activity rather than or in addition to oxygenase activity. One notable example is RPE65 which converts fatty acid all- trans retinyl esters (predominantly palmitate) into 11-cis-ROL via a concurrent isomerization and atypical hydrolysis reaction [29, 30, 86]. This enzyme plays an indispensable role in the regeneration pathway for 11-cis-retinal, the chromophore of retinal photoreceptor visual pigments. RPE65 loss-of-function mutations cause severe retinal dystrophies, such as retinitis pigmentosa and Leber congenital amaurosis (LCA), leading to blindness [67-69].

The CCO structural fold consists of a seven-bladed β-propeller capped on the top face by non-contiguous α helices and loops that together form the substrate binding tunnel. Residues lining this tunnel are generally hydrophobic and thus provide an ideal environment to accommodate apolar carotenoid substrates [12,

14, 15, 127]. Despite this shared physicochemical property, the tunnels observed in published CCO structures differ substantially in their shapes and amino acid

compositions. The geometric and steric restrictions imposed by the residues within this region are believed to underlie the stereo and regio-selectivity of CCO enzymes [74]. However, there are currently no published structures of genuine

CCO-substrate complexes that validate this hypothesis, as the generally poor aqueous solubility of carotenoids in general poses a formidable challenge to CCO structural studies [15, 127]. Although a few prior studies have examined the functional importance of active site residues in CCO substrate interactions, metal binding and catalytic activity [15, 36, 59, 60, 103, 128], detailed structural and biochemical studies concerning this issue are lacking.

The CCO iron catalytic center is positioned deep within the substrate binding tunnel with the Fe(II) cofactor coordinated by four strictly-conserved His residues, leaving one or potentially two open coordination sites, depending on the specific CCO, accessible for ligand binding. In addition, three conserved Glu residues indirectly contribute to iron coordination through hydrogen bonding interactions with three of the direct His ligands. This 4-His+3-Glu dual-sphere metal binding motif distinguishes CCOs from other non-heme, mononuclear iron centers

[129]. Involvement of the Glu-sphere is indispensable for CCO catalytic function as shown by previous studies [36, 86, 130]. Additionally, mutations in the RPE65 gene that cause substitutions in second sphere Glu residues are associated with severe retinal dystrophy [131]. Previous biochemical studies imply that the negative-charged carboxylate groups of these Glu residues are involved in iron charge neutralization [36, 130], but their precise role in maintaining the structure of the iron center and CCO catalytic activity remains elusive.

Synechocystis apocarotenoid oxygenase (ACO) is a prototypical CCO that is amenable to structure-function studies including crystallographic analysis of point mutants [12, 127, 132]. Here, we employed this enzyme as a model to probe the relationship between CCO active site structure, catalytic activity and regioselectivity. Our results indicate that while many ACO active site mutants have impaired catalytic activity, the regio-selectively of this enzyme is highly resistant to active site amino acid substitutions. Structural analysis of a particularly detrimental point mutant (W149A) revealed unexpected disruptions in regions of the active site distant from the where the Trp side chain normally resides. Two of these perturbed residues, H238 and E150, are involved in iron coordination, the latter residue being homologous to E148 in RPE65, a position at which Asp substitution has been associated with LCA [130, 131]. In both D150 and Q150 ACO point mutants the non-native side chain fails to form a fully stable interaction with H238, which in turn disrupts the H238-Fe(II) coordinate bond. This destabilization of the iron center likely contributes to the low catalytic activity of D/Q150 ACO mutants and the retinal pathology associated with the E148D variant of RPE65. Thus, in addition to providing key insights into the active site determinants of CCO catalysis, these data provide novel structural details regarding the role of second sphere Glu residues in iron coordination by CCOs and help illuminate the molecular pathology associated with an RPE65 point mutation.

3.2 Materials and Methods

3.2.1 In silico ligand docking

The substrate coordinate and stereochemical restraint files for all-trans-8’- apocarotenol were generated with the PRODRG server [133]. Adjustments to the polyene configuration and regularization of the substrate bond lengths and angles were performed using COOT [91]. The substrate coordinate file was then processed with Autodock Tools to generate a pdbqt file with polar hydrogens added to the ligand. The polyene single bonds were allowed to freely rotate.

Docking was accomplished with a 2 Å resolution ACO crystal structure (PDB ID,

4OU9) [127] in which water molecules in the model were removed. Autodock Tools were used to convert the model to a pdbqt format with polar hydrogens added in the protein model. Docking experiments of ACO with substrate were then carried out with Autodock Vina 1.1.2 [134]. Multiple hits were identified during the docking trials which were carried out using a search area that included the entire ACO active site. The top binding pose, in which the C15-C15’ double bond was placed in close proximity to the iron center with the β ionone ring positioned near the membrane binding surface of the protein was used to guide the mutagenesis study.

3.2.2 Molecular biology, protein expression and purification

All Synechocystis ACO point mutants were generated from a previously described pET3a-ACO expression plasmid [127] with a QuikChange site directed mutagenesis kit (Stratagene, Santa Clara, CA) and confirmed by DNA sequencing.

ACO was expressed as previously described with minor changes [127, 132]. The

LB culture supplemented with ampicillin (100 µg/ml) and ferrous iron (ammonium

ferrous iron sulfate or Mohr’s salt, 50 µg/ml) was grown at 37 ˚C, with 230 rpm

shaking to an OD600nm of ~ 0.6, and induced by adding IPTG to a final concentration of 100 µM. At the same time, additional ampicillin (100 µg/ml) and iron salt (50

µg/ml) were added into the culture. After an overnight incubation at 28 ˚C, cells were harvested by centrifugation and suspended in 20 mM HEPES-NaOH, pH 7.0.

Cells were either flash-frozen and stored at -80 ˚C or used immediately. ACO

purification was performed as previously described [127]. All ACO mutants were

expressed and purified identically to the wild-type protein. Concentrations of

purified ACO samples were determined using an A280 nm extinction coefficient of

75249 M-1·cm-1 as determined by amino acid analysis of purified wild-type ACO

(Protein Chemistry Laboratory, Texas A&M University).

3.2.3 Enzymatic assays, high performance liquid chromatography (HPLC), and

mass spectrometry (MS) analyses

Activity studies of ACO and mutants were performed by previously

established methods [127]. Briefly, 2 µg of purified ACO was added to 200 µl of

reaction buffer consisting of 20 mM HEPES-NaOH, pH 7.0, 0.05% (w/v) Triton X-

100, and 1 mM TCEP, pH 7.0. All-trans-8’-apocarotenol substrate in DMSO was

added to initiate the reaction. The reaction proceeded for 20 min at 28 ˚C with 500

rpm shaking in the dark and then was quenched with 200 µl methanol. Products

were extracted with 500 µl hexane, and the analysis was performed by HPLC on

a ZORBAX SIL (5 µm, 4.6 × 250 mm) normal phase column (Agilent, Santa Clara,

CA). 100 µl of extract were injected into the column and elution with hexane/ethyl

acetate (4:1, v/v) was carried out at 1.4 ml/min. RAL was quantified by plotting

peak areas of known quantities of an authentic standard (TRC, Toronto, Canada).

Enzymatic studies of all ACO mutants were conducted identically to those carried out with wild-type protein. For mass spectrometry analysis of products formed by wild-type and mutant forms of ACO, hexane extract (100 µL) from the enzyme reaction mixtures were injected into the HPLC system as described above and the eluate was directed into the atmospheric pressure chemical ionization probe source of an LXQ linear ion trap mass spectrometer (Thermo Scientific, Waltham,

WA). Mass spectra were analyzed with the Xcalibur 2.0.7 software package.

3.2.4 Determination of kinetic parameters

For analysis of steady-state kinetics, ACO-catalyzed reactions were performed in a 96-well plate (Thermo Fisher, Waltham, WA) with different all-trans-

8’-apocarotenol concentrations ranging from 0 to 150 µM. Inhibition of ACO activity was observed at substrate concentrations above 150 µM. 100 µl of enzyme solution containing 4 µg of purified protein in reaction buffer and substrate at various concentration in the same buffer were individually added to the plate wells.

The plate was incubated at 28 ˚C for 10 min before mixing the enzyme solution with substrate. Reaction progression was monitored with a Flexstation3 microplate reader (Molecular Devices, Sunnyvale, CA) by measuring the change in substrate absorbance at 424 nm over time. Reactions at each substrate concentration were performed in triplicate. Care was taken to include only the linear portion of the progress curves for initial velocity measurements. A standard curve made with known substrate concentrations was used to quantify substrate depletion. Analysis

of the kinetic data was performed with SigmaPlot (Systat Software, Inc., San Jose,

CA).

3.2.5 Protein crystallization, structural determination, and analysis

Crystallization of ACO mutant enzymes was performed as described previously for the wild-type protein [127, 132]. Briefly, purified protein samples were loaded onto a 25 ml Superdex 200 gel filtration column equilibrated with 20 mM HEPES-NaOH, pH 7.0, containing 0.02% (w/v) Triton X-100. A single, symmetrical peak that eluted at ~13 ml was collected and concentrated to 8 - 10 mg/ml. For crystallization, 1.5 - 2 µl of purified enzyme at 10 mg/ml in gel filtration buffer was mixed with reservoir solution containing 0.1 M BisTris propane-HCl, pH

6.0, 21–23% (w/v) sodium polyacrylate 2100, and 0.2 M NaCl in a 1:1 ratio.

Numerous trials yielded crystals with suboptimal morphology and poor diffraction quality. To improve crystal quality, a microseeding method was applied to obtain crystals of each mutant ACO. Specifically, after mixing protein samples with reservoir solution a small quantity of crushed wild-type ACO microcrystals were applied to each drop. Crystallization was carried out by the hanging-drop vapor- diffusion method at 8 °C. Rod-shaped crystals typically appeared within 2 - 3 weeks. Mature crystals were directly harvested and flash-cooled in liquid nitrogen before X-ray exposure. Diffraction data were collected at the NE-CAT 24-ID-E beamline of the Advanced Proton Source and indexed, integrated, and scaled with the XDS package [89]. Mutant ACO crystals were isomorphous to previously reported orthorhombic wild-type ACO crystals, and their structures were determined by rigid body refinement in REFMAC5 with PDB entry 4OU9 used as

the starting model [92]. Manual adjustments to the structure were made with COOT

[91], and restrained refinement was carried out in REFMAC5 [92]. Structures were validated with MOLPROBITY [95] and the wwPDB structure validation server [135].

In crystals of each mutant ACO, a fifth, less well-defined ACO molecule was identified within the asymmetric unit. The X-ray data and refinement statistics are summarized in Table 4.3. All structural figures were prepared with PyMOL

(Schrödinger, New York, NY).

3.3 Results

3.3.1 Identification of potential substrate-interacting residues for mutagenesis studies

ACO recognizes C25-35 β-apocarotenals/ols and specifically cleaves them at the C15-C15’ double bond to form RAL and a second linear apocarotenal product

(Fig. 3.1A) [45]. Among different length β-apocarotenoids, ACO displays highest activity towards all-trans-8’-apocarotenol and its 3-hydroxy derivative. The former is commercially available, making it convenient for activity studies (Fig. 3.1A). To identify active site residues important for substrate recognition and cleavage site selectivity, we carried out in silico docking studies of all-trans-8’-apocarotenol in the ACO active site. In multiple docking runs the top-binding pose oriented the substrate with the β-ionone moiety interacting with residues at the entrance of the binding pocket and the C15-C15’ double bond in close proximity to the iron center

(Fig. 3.1B). Three residues with bulky aromatic side chain (W149, F113, F236) were proposed to control the depth of substrate entry into the active site entry such that the 15-15’ double bond is appropriately positioned close to the iron center to

facilitate efficient and regioselective cleavage (Fig. 3.1B) [12].The scissile bond was surrounded by the phenyl side chains of F69 and F303 (Fig. 3.1B). Based on their proximity to the site of cleavage we reasoned that they could reinforce cleavage selectivity as well as help stabilize reaction intermediates. In the innermost region of the cavity, three residues (Y24, L400, F371) appeared to help form a pocket that could accommodate the distal polar end of the substrate. As the only residue containing a polar hydroxyl moiety, Y24 may form a direct or water- mediated interaction with the substrate hydroxyl tail that is located 5.7 Å away in the docked model (Fig. 3.1B). Overall, the substrate docking pose is consistent with the known biochemical properties of ACO. Therefore, this enzyme substrate model was used to guide our mutagenesis studies. We divided the abovementioned residues into three groups based on their locations: the β-ionone ring binding region (W149, F113, F236), the region flanking the cleavage site (F69,

F303), and the distal end in the binding pocket (Y24, F371, L400) (Table 3.1).

3.3.2 ACO active site mutants impair catalytic activity to various degrees without altering regioselectivity

Three residues located in the β-ionone binding region (F113, F236, W149) and two in the distal pocket (F371, L400) were substituted with alanine to probe their contributions to substrate interactions. Y24 also was mutated to a phenylalanine (Y24F) to examine its potential to hydrogen bond with the substrate’s hydroxyl group. Phe residues 69 and 303, which flank the scissile double bond were replaced either independently with alanine (F69A, F303A) or together (F69A/F303A) to evaluate their involvement in regioselectivity and overall

catalytic activity. The effect of introducing a polar functional group into the generally apolar environment that surrounds the catalytic center was examined with F69Y substituted ACO. Mutants examined in this study are summarized in

Table 3.1.

All ACO mutants were expressed and purified in the same manner as wild- type protein. The remaining mutants featured detectable but variable levels of expression compared to wild-type ACO (Table 3.2). SDS-PAGE analysis of each purified ACO showed a similar homogeneity, with the exception of F69Y which was markedly diminished (Fig. 3.2A) consistent with its lower expression level compared with the other mutants (Table 3.2). Surprisingly, except for F69Y, oxidative cleavage activity as assessed at a single substrate concentration by

HPLC was retained by all mutants (Fig. 3.2B). Among them, five ACO mutants

(F69A, W149A, E150Q, F371A and L400A) showed < 5% activity, whereas the others displayed enzymatic activity ranging from 8% to 76% of the native protein

(Fig. 3.2B).

To test possible changes in regioselectivity, we analyzed the reaction products by HPLC-MS. Whereas all-trans-retinal was the only β-apocarotenoid product of wild-type ACO detectable by UV/Vis absorbance profiles, the atypical all-trans-12’-apocarotenal product of oxidative cleavage at the C11’-C12’ position was detected by HPLC-MS (Fig. 3.3A and B). Each single mutant also produced

RAL as the dominant product (Fig. 3.2B), indicating an unaltered selectivity for the cleavage site. Products derived from the double mutant F69A/F303A were also analyzed to assess whether the reduced steric hindrance in vicinity of the cleavage

site in this mutant protein (Fig. 3.1A) could potentially render a more pronounced change in regioselectivity. Indeed, production of all-trans-12’-apocarotenal was

increased ~2-fold in this double mutant compared to wild-type protein although the

quantity produced was still below the limits of UV/Vis detection (Fig. 3.3A and B).

These results demonstrated that active site mutants differentially affected catalytic

activity, but that regioselectivity of ACO is preserved in the face of diverse active

site perturbations.

3.3.3 Active site mutants primarily affect maximal enzymatic activity rather than

the Michaelis constant

To further investigate the enzymology underlying the impaired activities

steady-state kinetic studies for each mutant were performed. Having established

the cleavage selectivity of each ACO variant by HPLC, we employed a more rapid

assay system for these steady-state kinetic studies in which substrate

consumption is monitored spectrophotometrically in a plate reader. The steady-

state kinetic parameters of native ACO determined by this method were in good

agreement with those obtained previously by HPLC analysis [127], thus validating

the assay methodology (Fig. 3.4). A majority of mutants showed only minimally

perturbed Km values (Y24F, F69Y, W149A, E150Q, F236A, F303A, L400A), with four variants (F69A, F113A, E150D, F69A/F303A) displaying ~2-3 fold increases

as compared to native ACO (Table 3.2 and Fig. 3.4). By contrast, kcat values for

most mutants were found to be substantially impaired. Y24F was the only variant

with kinetic constants similar to the native enzyme, suggesting a less critical role

for this residue in catalysis compared to the others examined (Table 3.2). Three

100

variants (F113A, E150D, F236A) had kcat values reduced by 70-80% with the remainder displaying maximal activities less than 10% of native protein (Fig. 3.4 and Table 3.2). In this assay system F69Y ACO exhibited some residual activity, whereas F69A, W149A, F371A and L400A ACO had specificity constants (kcat/Km) less than 3% of wild-type enzyme (Fig. 3.2A and B).The drastically decreased kcat values for many of the ACO variants examined implied important roles in maintenance of catalytic efficiency for residues in all regions of the active site pocket. The less pronounced effects of most substitutions on the stability of the enzyme-substrate complex at steady-state (i.e. the Km value) together with the preserved regioselectivity of the variants indicate that residues lining the substrate binding tunnel work in concert to create a rigid platform for substrate binding and processing.

3.3.4 The crystal structure of W149A ACO reveals major disruptions in the substrate-binding cleft and metal coordination

The bulky hydrophobic side chain of W149 and its proximity to the β-ionone ring of the substrate suggest an important function for this residue in substrate interactions (Fig. 3.1B). The major reduction in activity of the W149A ACO variant

(Fig. 3.2B), prompted us to investigate the underlying mechanism of this catalytic inactivation. To this end, W149A ACO was crystallized under the same conditions used for native ACO. The best crystals diffracted to ~ 2.8 Å resolution and were isomorphous to the previously reported orthorhombic ACO crystals [13]. In this

ACO mutant structure as well as those discussed below we observed electron density in the solvent region consistent with the presence of a fifth ACO molecule

101

in the asymmetric unit. The extra protomer was modeled in the final structure but was not used to draw mechanistic conclusions due to its weak electron density relative to the other modeled chains. Superimposition of W149A and native ACO revealed an average RMSD of ~ 0.425 Å between Cα atoms. Absence of electron density for the W149 side chain confirmed the Ala substitution (Fig. 3.5B).

Despite the overall structural similarity to native ACO, inspection of the

W149A mutant catalytic center revealed an unexpected disruption in the iron coordination center (Fig. 3.5A compared to 5B). E150, a member of the conserved

3-Glu second sphere iron binding motif adjacent in sequence to the mutated site, adopted an alternative conformation that abolished its normal hydrogen bonding interaction with H238. This change was accompanied by enhanced mobility of the

H238 side chain as evidenced by its weakened electron density as well as a loss of the coordinate bonding interaction with the iron cofactor (Fig. 3.5B), resulting in a 3-coordinate iron center. The perturbation in iron-coordination resulted in an elevated iron B-factor (96 Å2 versus 31 Å2 in native ACO at a comparable resolution), which reflects increased iron mobility and/or reduced occupancy within the binding site. These structural perturbations could be directly attributed to alterations in the local structure surrounding position 149. W149 and E150 are located in the i and i+1 positions of a type 1 β turn connecting the inner strands of blade 1 of the β propeller fold. Trp is infrequently found at the start of such β turns

[136] and the bulky indole moiety could help enforce the proper geometry of E150.

Notably, the ψ angle of -9.3º for E150 differs significantly from the ideal value of -

30º for an i+1 residue in a type 1 β turn possibly as a consequence of steric effects

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resulting from the bulky Trp side chain [137]. The Ala substitution at position 149

converts the β turn into a type IV conformation with loss of a hydrogen bonding

interaction between the i and i+3 residues. These changes are accompanied by a

~2 Å shift in the E150 Cα atom. Together, these structural alterations change the

environment of the E150 side chain such that its interaction with H238 is no longer

favored. In addition to alterations in the iron center, electron density for F236 which

is adjacent to H238 was also substantially weakened indicating an enhanced

mobility likely resulting from the H238 structural perturbation (Fig. 3.5B).

Interestingly, despite its extremely low activity, W149A displayed a Km similar to that of native ACO, indicating a preserved substrate binding capacity. Because

F236A exhibited an activity and enzymatic parameters comparable to native ACO, inactivation of W149A could be linked to disruption of the iron center and, to a lesser extent, a deficiency in substrate binding.

3.3.5 E150 in the second sphere is critical for metal binding, maintenance of active

site structure and catalytic activity

Our results strongly suggested a critical role for E150 in iron coordination

and ACO catalytic function. To directly assess its structural and functional role in

iron coordination, E150 was substituted with Asp, which has a one methylene

shorter side chain and thus is likely incapable, within the confines of the β turn

structure, of hydrogen bonding with H238. Since an equivalent substitution in

RPE65 (E148D) was found in patients with LCA, characterization of this mutant

would improve our understanding of RPE65-associated retinal pathology.

Whereas the expression and purification of E150D resembled that of native ACO,

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its maximal catalytic activity was reduced by ~ 80% with a 2-3 fold increased Km value (Fig. 3.2A and B, Table 3.2). To examine the structural consequences of this substitution, we determined the crystal structure of the mutant protein at a resolution of ~ 2.8 Å. Like the W149A mutant structure discussed above, the

E150D crystals contained a fifth monomer in the ASU albeit with weaker electron density support compared to the other chains. Electron density for D150 was clearly evident, but indicated that the side chain was flipped to a vacant site opposite the iron center (Fig. 3.6A). The hydrogen bonding interaction between

E150 and H238 was eliminated, resulting in disruption of the coordination between

H238 and iron. Interestingly, H238 is well-resolved but its side chain points away from the iron (Fig. 3.6A), clearly demonstrating an iron coordination defect in

E150D ACO. Consequently, the altered three-coordinate metal center featured a reduced iron occupancy and/or high mobility as evidenced by an elevated iron B- factor. Like in W149A ACO, F236 exhibited a high mobility manifested as poorly resolved side chain electron density (Fig. 3.6A).

Previous mutagenesis studies of RPE65 suggest that the negative charge of carboxylate groups from the 3-Glu sphere contribute to iron charge neutralization [36, 130]. It has also been suggested that the second sphere Glu residues help fix the first shell His side chains in conformations capable of stably coordinating iron. To test these hypotheses, we replaced E150 with Gln, which contains an uncharged side that maintains a capacity to hydrogen bond with H238.

This substitution yielded a nearly inactive enzyme (Table 3.2 and Fig. 3.4D). The crystal structure of E150Q ACO solved at 2.75 Å resolution, revealed changes in

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the catalytic center largely resembling those seen in the W149A and E150D ACO mutants, including loss of coordinate bonding between H238 and the iron cofactor, an elevated iron B factor and increased mobility of F236. However, unlike E150D

ACO, the Gln residue maintained a hydrogen bonding interaction with H238 (Fig.

3.6B). Indeed, the electron density for the H238 imidazole ring is more discernable in E148Q ACO than that in the W149A and E150D mutants. However, coordination of H238 to the iron was clearly disrupted, as demonstrated by the average ~ 3.4 Å distance separating the H238-Nε and iron atoms (Fig. 3.6B).

3.4 Discussion

The biological functions of CCOs have been described in numerous studies.

However, uncertainties remain regarding how CCOs interact at the molecular level with carotenoids to determine their substrate specify as well as their regio- and stereo-selectivity. With those questions in mind, we employed a cyanobacterial

ACO as a model for the CCO family to systematically examine the functional and structural importance of its active site resides. As revealed by our computational docking model, the steric influence exerted by three hydrophobic residues (W149,

F113, F236) functions as a bottleneck that prevents further passage of the β- ionone moiety of the apocarotenoid molecule into enzyme’s active site. This model agrees with a previous representation of the ACO-substrate complex proposed by

Kloer et al [12]. Models in which the substrate orients with the β-ionone ring residing in the interior region of the tunnel were ruled out based on the inability of

ACO to cleave β-carotene [45]. Rather the bottleneck hypothesis is consistent with experimental data revealing that ACO can accept apocarotenoid substrates with

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polyene backbones of various lengths but only cleaves them at the C15-C15’ position [45] (Fig. 4.1).

Among the residues examined in this study, the position homologous to

F236 in ACO has also been studied in mouse BCO1 (Y235) and maize CCO1

(M345) [15, 138]. Interestingly, Y235 in BCO1 was hypothesized to stabilize a proposed cationic [138] reaction intermediate that is believed to occur commonly in CCO-catalyzed oxidative reactions [34]. This was supported by the fact that substitutions of Y235 with aromatic residues (Y235F/W) that minimally perturbed

BCO1 activity, whereas non-aromatic amino acid replacements such as Y235L led to ~ 50% activity reduction [138]. Indeed, a similar carbocation stabilization mechanism has been proposed for RPE65 [60] and some isoprenoid-metabolizing enzymes [139, 140]. In contrast to BCO1, the F236A substitution in ACO and the homologous mutant M276A in CCO1 failed to exhibit significant activity loss [15], which argues against a role for F236 in carbocation stabilization, at least in ACO.

CCO sequence alignments and structural superpositioning suggest that a consensus Phe residue may fulfill this function. This conserved Phe residue (F69 in ACO, F171 in VP14, and F61 in RPE65) is located across from the iron center near the predicted position of the scissile double bond [13-15] where it could play a key role in stabilizing reaction intermediates.

Changes in substrate regio- and stereo-selectivity in response to active site alterations have been demonstrated in several dioxygenases, e.g. naphthalene and toluene dioxygenases [141-143]. Inspired by these studies, we first analyzed the reaction products of native ACO and identified an atypical apocarotenoid

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product formed by cleavage at the C11’-C12’ site. However, the trace amount of this atypical product, detectable only by mass spectrometry, indicated that ACO- catalyzed polyene cleavage is tightly regulated with a high degree of precision (Fig.

3.3). We hypothesized that mutation of a substrate-interacting residue could enhance formation of the atypical product. Surprisingly, all tested ACO mutants faithfully generated RAL as the dominant product (Fig. 3.2B), with only a minor increase in formation of the atypical product observed for the F69A/F303A dual- mutant (Fig. 3.3). These data suggest that residues in the substrate binding cleft act together in substrate binding and processing, such that regioselective cleavage is maintained in the presence of subtle active site changes. However, formation of the atypical product could be underrepresented in our analyses, as it could be further processed by the enzyme with RAL as the final product.

Conserved second sphere Glu and Asp residues are frequently found in non-heme iron enzymes. In 15-lipoxygenase, for instance, Glu357 in the outer iron sphere is critical to enzyme’s activity as well as reaction specificity [144, 145].

However, CCOs appear to employ the most extensive use of a negatively charged second ligand sphere for maintenance of the iron center structure and function amongst this class of enzymes. The strict conservation of Glu, or in rare cases Asp, at the second sphere positions in this ancient family indicates a profound selective pressure maintaining the anionicity that envelops the 4-His Fe(II) center [40, 129].

The neutral 4-His inner sphere of CCOs contrasts with metal-binding ligand sets of most other non-heme iron dioxygenases, which usually contain at least one negatively-charged residue [42]. The non-heme iron of photosystem II constitutes

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the only other known example of a 4-His-Fe(II) center lacking protein-associated negatively charged iron ligands [44]. Therefore, the neutral CCO 4-His combination could be insufficient to cage the iron thus requiring outer sphere anionic ligand(s) to achieve a stable coordination complex. The ability of Dke1 [146] and cysteine dioxygenase [147] to complex Fe(II) with a 3-His triad motif somewhat argues against this hypothesis although these enzymes also contain Fe-bound solvent molecules that may contribute to charge stabilization. By contrast, most CCO iron centers are likely capable of binding only a single solvent molecule [74, 132]. The presence of an anionic Glu side chain in close proximity to the His ligands is expected to elevate the pKa of the inner sphere imidazole rings [148], which in turn would stabilize the His-Nε-iron coordinate bond and tune the reactivity of Fe(II) towards dioxygen. The disrupted interaction between E150 and H238 in W149A

ACO along with severe defects in the catalytic center coupled with almost zero activity emphasize the significance of the 3-Glu sphere.

To further probe the role of E150 in ACO function, two point mutants, E150D and E150Q, were structurally and kinetically characterized. In E150D ACO, the loss of iron coordination by H238 together with a ~3.3-fold reduced kcat unambiguously demonstrated a critical role of Glu in iron coordination and catalytic function. The structure of E150Q ACO revealed that this non-native residue, like

Glu, forms a hydrogen bonding interaction with H238, similar to that of the native

Glu side chain. However, this interaction did not promote formation of a stable coordinate bond between H238 and the iron cofactor. Interestingly, E150Q ACO was essentially inactive despite being expressed at a level comparable to the wild-

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type ACO suggesting that the Gln side chain, per se, exerts an additional negative effect on the catalytic properties of this enzyme. These results are consistent with previous studies showing that Gln substitution for one of the conserved outer sphere Glu residues in RPE65 was substantially more detrimental for catalytic activity than an Asp substitution [36, 130]. It is conceivable that the D150 ACO variant could partially stabilize H238 in a catalytically competent orientation, particularly in the presence of substrate, to enable the observed residual activity.

Indeed, in the crystal structure of E150D, weak electron density was consistently observed between the iron cofactor and H238, which suggests that the ensemble of His conformations may include the native rotamer and that this subpopulation may be responsible for the residual activity. Cumulatively, our results reinforce the notion that the negativity of the 3-Glu motif is indispensable for CCO catalysis.

In addition to a role in iron coordination, a recent crystal structure of RPE65 complexed with emixustat, a retinoid-mimetic inhibitory amine, disclosed a role for

Glu148 in retinyl cation stabilization [149]. In this high resolution crystal structure, the Glu148-Oε atom is located 2.9 Å from the amine group of the compound, which suggests its high tendency to form an electrostatic interaction with the C15 retinyl carbocation during catalysis [149]. In the calculated ACO-substrate model, the distance between the C15 site in the substrate and oxygen atom of E150 is ~ 6 Å.

Electrostatic contacts could be formed between E150 and cationic apocarotenoid intermediates, but further structural and biochemical studies are needed to determine if such a role is fulfilled by this residue in ACO.

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In summary, our study provides a systematic mutagenesis approach targeting active site residues that are potentially involved in carotenoid interactions.

Detailed biochemical and kinetic data obtained with purified enzymes allowed us to test the functions of these residues in substrate interactions. In addition, our crystallographic and kinetic studies on selected ACO mutants provided insights needed to understand the unusual 4-His+3-Glu iron-coordination system in CCOs, particularly the 3-Glu outer sphere. As a consequence of our studies with ACO, we also provide a plausible molecular mechanism underlying the functional impairment of an RPE65 mutant associated with human retinal dystrophy.

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Tables

Table 3.1. ACO mutations and their corresponding locations in the active site relative to the docked substrate ligand.

Mutation sites Targeted Mutants

residues

β-ionone proximal region of the F113, W149, F113A, W149A, F236A

substrate F236

Flanking the substrate scissile F69, F303 F69Y, F303A,

double bond F69A/F303A

Distal end in the binding tunnel Y24, F371, Y24F, F371A, L400A

interior site L400

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Table 3.2. Steady-state kinetic constants for wild-type and active site- substituted ACOs

b ACO Mutant Expression Km Vmax kcat kcat/Km RPE65 Disease

level (µM) a (µM/m in) (min-1) (min-1 equivalent

µM-1)

WT ++++ 23.9 ± 6.36 ± 17.26 ± 0.722 ± --

4.9 0.41 1.11 0.155

Y24F ++ 27.3 ± 5.66 ± 15.36 ± 0.563 ± F16 Polymorphism

5.8 0.39 1.06 0.126

F69A + 66.8 ± 0.22 ± 0.60 ± 0.008 ± F61

9.9 0.03 0.08 0.002

F69Y ++ 21.6 ± 0.06 ± 0.65 ± 0.030 ± F61

4.3 0.01 0.12 0.008

F113A ++ 47.6 ± 1.15 ± 3.12 ± 0.066 ± F103

19.7 0.19 0.52 0.029

W149A ++ 29.6 ± 0.09 ± 0.24 ± 0.008 ± T147

5.4 0.01 0.02 0.002

E150D +++ 79.9 ± 1.88 ± 5.10 ± 0.064 ± E148 LCA c

7.7 0.09 0.24 0.007

E150Q +++ 21.8 ± 0.07 ± 0.19 ± 0.009 ± E148

3.7 0.01 0.03 0.002

F236A +++ 21.2 ± 2.22 ± 6.03 ± 0.284 ± Y239 LCA

3.6 0.13 0.35 0.051

F303A ++ 23.8 ± 0.67 ± 1.82 ± 0.076 ± F312

4.3 0.04 0.11 0.015

F371A + 22.0 ± 0.07 ± 0.19 ± 0.009 ± F418

1.5 0.01 0.27 0.012

L400A + 26.3 ± 0.23 ± 0.62 ± 0.024 ± P444

2.2 0.01 0.03 0.002

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F69A/F303A + 50.4 ± 0.62 ± 1.68 ± 0.033 ± F61/F312

9.2 0.05 0.14 0.007

a Kinetic data were obtained with purified recombinant proteins b Disease caused by mutation of the homologous position in RPE65. c Leber congenital amaurosis

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Table 3.3. X-ray crystallographic data collection and refinement statistics for

ACO mutants

Data collection a

Crystal name W149A ACO E150D ACO E150Q ACO

Beamline NECAT 24-ID-E

Wavelength (Å) 0.97921 0.97910 0.97919

Space group P212121

Unit cell a = 118.14 a = 118.42 a = 118.53

parameters (Å) b = 125.26 b = 124.95 b = 125.50

c = 203.57 c = 203.97 c = 203.60

Resolution (Å) 48.62-2.80 48.58 – 2.81 (2.88 47.42 – 2.75 (2.82

(2.87-2.80) a – 2.81) a – 2.75) a

Unique reflections 75,013 (5,505) 74,574 (5384) 75,683 (3786)

Completeness (%) 100.0 (100.0) 99.9 (98.6) 95.0 (65.1)

Multiplicity 8.2 (8.1) 8.1 (7.9) 3.8 (3.1)

11.0 (1.1) 7.9 (1.1) 7.6 (1.4)

RmeasI (%)b 20.3 (217.9) 23.4 (172.5) 20.0 (124.4)

CC1/2 (%)b 99.5 (30.5) 99.1 (49.6) 98.5 (33.7)

Refinement

Resolution (Å) 48.62 – 2.80 48.58 – 2.81 47.42 – 2.75

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No observations 71,417 70,990 72,122

Rw ork/Rfree (%)c 19.8/23.9 20.2/23.3 21.2/24.7

No atoms 18,975 18,909 18,952

Protein 18,814 18,835 18,840

Water 154 69 107

Metal/ion 5 Fe, 2 Cl 5 Fe 5 Fe

B-factors (Å2)

Protein 76 73 71

Water 42 39 35

Metal/ion 107 (Fe), 59 (Cl) 99 (Fe) 89 (Fe)

RMS deviations

Bond lengths (Å) 0.01 0.01 0.01

Bond angles (°) 1.38 1.33 1.27

Ramachandran plotd

Favored/outliers 97/0 97/0 96/0

(%)

PDB accession 5KJA 5KJB 5KJD

code a Values in parentheses are those for the highest resolution shell of data b As calculated in XDS

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c As calculated in REFMAC d As calculated in MOLPROBITY

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Figures

Figure 3.1. ACO-catalyzed cleavage reaction and model of the ACO-substrate complex. A. ACO accepts and specifically cleaves all-trans-8’-apocarotenol at the

C15-C15’ double bond (red wavy line) with incorporation of both O2-originated oxygen atoms into the RAL and C10-aldehyde apocarotenoid products. B. Stick view of an in silico docking model of ACO (PDB ID: 4OU8) complexed with all- trans-8’-apocarotenol. The most energetically favorable binding pose is featured with residues located close to the docked apocarotenoid molecule (in grey) shown as orange sticks. The iron cofactor, shown as a brown sphere, is close to the scissile C15-C15’ double bond (labeled as C15).

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Figure 3.1.

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Figure 3.2. Expression, purification and activity of native and mutant ACOs.

A. SDS-PAGE analysis of wildtype and ACO mutants purified by (NH4)2SO4 fractionation and gel-filtration chromatography. Proteins were separated on a 4 –

20% BisTris/glycine gradient gel and visualized by Coomassie R-250 staining. The arrow on the right indicates the position where ACO migrates. Note that most ACO mutants were purified to levels comparable to that of the wild-type protein. B.

Activities of wild-type and mutant ACOs. Activity was quantified by HPLC monitoring of RAL formation from the all-trans-8’-apocarotenol substrate. Error bars represent standard deviations computed from duplicate results of a single experiment.

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Figure 3.2.

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Figure 3.3. Formation of all-trans-12’-apocarotenal by native and

F69A_F303A ACO. A. Detection of all-trans-12’-apocarotenal product formation by mass spectrometry. Extracted ion chromatogram of the reaction products generated by F69A_F303A showing elution of an m/z 351.2 ion intensity peak at

~ 4.8 min. B. MS analysis of the product signal in panel A showing a dominant ion at m/z 351.19, consistent with the protonated form of all-trans-12’-apocarotenal generated by cleavage at the C11’-C12’ double bond of all-trans-8’-apocarotenol.

Tandem MS analysis of the m/z 351.19 peak revealed a major fragmentation product at m/z 333.3 representing loss of water from the parent ion. C. Semi- quantification of the atypical product formed by mutant and wild-type ACO according to the peak intensities in panel A. Formation of the atypical product was a linear function of the amount of enzyme in the reaction mixture up to a mass of

20 µg. Error bars represent standard deviations from experiments performed in duplicate.

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Figure 3.3.

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Figure 3.4. Steady-state kinetics of native ACO and mutant proteins. ACO mutants were divided into four groups based on their location with respect to the apocarotenoid molecule (see Table 3.1 for details). Panels on the right in A through

D highlight the location of selected residues (in orange) in relationship to the docked substrate molecule (in grey). Steady-state kinetic plots for the highlighted mutants are shown in the panels on the right. Kinetic measurements were repeated at least twice using freshly purified enzymes and the results were comparable for each ACO sample. The derived steady-state kinetic parameters are summarized in Table 3.2.

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Figure 3.4

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Figure 3.5. Alterations in the iron center and active site cavity observed in

W149A ACO crystal structures. A. Iron center environment in native ACO (PDB

ID: 4OU8). The iron is coordinated by 4-His residues. The hydrogen bond between

E150, a member of the conserved 3-Glu sphere, and H238 is evidenced by their

close proximity and connected electron density. Note the well-resolved density

signal for the protein and the iron metal. B. Active site structure of W149A ACO.

The absence of side chain electron density near position 149 confirmed this

mutation. Note that E150 exhibits a well-discerned side chain signal but with a

rotameric conformation different than that in the native structure shown in panel A,

which abolishes the hydrogen bond between E150 and H238. The absence of a

defined electron density for the F236 side chain indicates increased mobility for

this region of the protein. In each panel the iron cofactor is shown as an orange

sphere and blue mesh represents the final σA-weighted 2Fo-Fc map contoured at

1 σ. Structures are presented as walleye stereo-pairs in each panel. Only residues

of interest are labeled in each panel.

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Figure 3.5.

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Figure 3.6. Disruptions of the active structure and iron coordination center in E150D and E150Q ACO. A and B. Active center structures of E150D (panel A) and E150Q (panel B) ACOs. Electron density of the non-native side chains are well-resolved in both structures. Compared to the W149A ACO structure, electron density for H238 is better defined in both E150 mutant ACO structures shown in

Figure 3.5, panel B. F236 is conformationally destabilized in both E150 mutant

ACO structures similar to what is observed in the W149A mutant structure.

Although the hydrogen bonding interaction between Gln150 and H238 is partially preserved in the E150Q ACO structure, H238 fails to form a normal coordinate bond with iron as evidenced by the lack of electron density signal between the two moieties. The red sphere and blue mesh represent the iron metal and the σA- weighted 2Fo-Fc map as in Figure 3.5 with residues of interest labeled in each panel. Structures are presented as walleye stereo-pairs in each panel.

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Figure 3.6.

128

CHAPTER 4: UTILIZATION OF DIOXYGEN BY CAROTENOID CLEAVAGE

OXYGENASES

This section was previously published in:

Sui, Xuewu, Marcin Golczak, Jianye Zhang, Katie A. Kleinberg, Johannes Von

Lintig, Krzysztof Palczewski, and Philip D. Kiser. "Utilization of dioxygen by carotenoid cleavage oxygenases." Journal of Biological Chemistry 290, (2015):

30212-30223.

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4.1 Introduction and background

Carotenoid cleavage oxygenases (CCOs) are non-heme iron-dependent enzymes found in all kingdoms of life that participate in the metabolism of carotenoids and related compounds. Since their initial discovery in bacteria and plants, tremendous progress has been made in elucidating the biological substrates of these enzymes [13, 150, 151]. Most characterized CCOs catalyze the oxidative cleavage of carotenoid carbon-carbon double bonds to yield aldehyde and/or ketone-containing apocarotenoid products (Fig. 4.1). Such activity was possibly the earliest to evolve in this family since apocarotenoids, particularly retinal, mediate photoreception by the ancient type 1 opsin protein family, members of which carry out many important physiological processes in phototactic and phototrophic organisms [152, 153]. Another group of bacterial and fungal members has evolved to cleave carbon double bonds of stilbenes such as resveratrol and lignin-related phenylpropanoids [154, 155]. The RPE65 class of

CCOs, found only in vertebrates, underwent catalytic neofunctionalization during its split from a beta-carotene oxygenase (BCO)-2-like ancestor to acquire the non- oxidative retinyl ester cleavage/isomerase activity central to the visual cycle that generates 11-cis-retinal required for visual pigment (type II opsin) formation [105,

156] (Fig. 4.1A).

The spin-forbidden reaction of O2 with alkene substrates of CCOs is made kinetically favorable through its reductive activation by the non-heme iron (II) centers of these enzymes [157]. Of central importance to understanding the mechanism of O2 activation by CCOs and its reaction with substrates is knowledge

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of whether one or both of the oxygen atoms from O2 is/are incorporated into the

reaction products. In the former case, the non-O2 derived oxygen atom found in the reaction is expected to originate from an active site-bound water molecule. A

number of studies using isotopically labeled O2 and H2O were carried out to

address this question for CCOs, but the issue still remains debatable [123, 158-

161]. A factor complicating the interpretation of such experiments is the exchange

of oxygen incorporated into the nascent products with that of bulk water during the

sample workup and analysis. This makes dioxygenases appear to be

monooxygenases [162, 163]. An initial study on the catalytic mechanism of chicken

BCO1 that suggested it was a monooxygenase [158] was criticized for its use of

conditions that allowed substantial solvent back-exchange [163]. More recent

studies of human BCO1 [159] and Arabidopsis CCD1 [160], in which solvent

exchange was carefully monitored, demonstrated dioxygenase labeling patterns

for these CCOs. By contrast, labeling experiments performed on resveratrol and

isoeugenol-cleaving CCOs, NOV2 [123] and IEM [161], from the non-

carotenogenic bacteria Novosphingobium aromaticivorans and Pseudomonas

nitroreducens, respectively, suggested a monooxygenase pattern of oxygen

incorporation for both enzymes. Thus, the labeling studies performed to date seem

to indicate that perhaps both monooxygenase and dioxygenase catalytic

mechanisms may be utilized within the CCO enzyme family [164], a possibility

previously suggested by a theoretical study of the catalytic mechanism of

Synechocystis ACO [34].

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In contrast to oxidative carotenoid cleavage by CCOs, a basic consideration

of the ester cleavage/isomerization chemistry catalyzed by RPE65 does not

suggest an obvious role for O2 in the retinoid isomerization reaction (Fig. 4.1B).

Additionally, substrate and water isotope labeling studies do not indicate direct

incorporation of O2-derived oxygen into the 11-cis-retinol product of the

isomerization reaction [14, 60, 84, 165]. However, the close structural similarity of

the RPE65 and carotenoid-cleaving CCO Fe(II) centers, together with the

evolutionary relatedness of these proteins, raise the possibility that certain features

of their catalytic modes could be shared including a requirement for O2 [74].

Recently, a hypothetical role for O2 in RPE65 catalyzed retinoid isomerization that does not involve its permanent incorporation into the reaction products has been proposed [166].

In the present study, we carried out isotope labeling studies on highly

purified and active preparations of Synechocystis ACO and Novosphingobium

NOV2 with assay protocols that minimized solvent oxygen back-exchange. These

experiments unambiguously showed that both enzymes, like human BCO1 and

Arabidopsis CCD1, are dioxygenases. A T136A substitution in the ACO sequence

that was predicted to open an occluded coordination site for solvent binding, thus

potentially favoring monooxygenase chemistry, did not alter the dioxygenase

labeling pattern [34]. Using ACO as an internal control, we demonstrated that O2

is not required for RPE65-mediated conversion of all-trans-retinyl esters into 11-

cis-retinol, in agreement with a recently proposed mechanism of retinoid

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isomerization [167]. These data provide compelling evidence for a common dioxygenase mechanism amongst all double bond-cleaving CCOs.

4.2 Materials and methods

4.2.1 Phylogeny inference

The CCO amino acid sequences of interest were retrieved from the NCBI protein database. Atomic coordinates for CCOs of known structure, Synechocystis

ACO, bovine RPE65, and Zea mays VP14 (PDB accession codes, 4OU9, 4RSC and 3NPE, respectively) were obtained from the Protein Data Bank. A structure- based sequence alignment was first generated using the program MUSTANG

[168]. This structure-based alignment was then used as a profile for sequence- based alignment of the remaining proteins using MUSCLE [169]. A standard sequence-based alignment of the proteins was also generated with MUSCLE to assess the influence of the alignment methodology on the inferred tree topology.

Columns containing gaps were removed from the alignment. The CCO phylogeny was inferred based on the maximum likelihood optimality criterion as implemented in PhyML [170]. The LG substitution matrix with an estimation of the gamma parameter (4 categories) and invariant sites resulted in the highest log-likelihood tree as assessed by ProtTest [171]. Improvements to the starting neighbor-joining tree were carried out by both nearest neighbor interchange (NNI) and surface pruning and regrafting (SPR) rearrangements. Both tree topology and branch lengths were optimized. Tree robustness was assessed by analysis of 1000 bootstrap pseudo-replicates. The presented maximum-likelihood tree had a log

133

likelihood value of -14321.6 (Fig. 4.1A). An identical tree topology was obtained when the procedure was repeated with the pure sequence-based alignment.

4.2.2 Protein expression and purification

Synechocystis ACO was expressed and purified as previously described

[127]. The ACO T136A point mutant was generated with a QuikChange Site-

Directed Mutagenesis Kit (Stratagene, Santa Clara, CA) and confirmed by DNA sequencing. T136A-ACO was expressed and purified identically to the wild-type protein. The coding sequence of NOV2 from Novosphingobium aromaticivorans

DSM 12444 (GI: 499765715) was synthesized and cloned into the pET3a expression vector (Novagen) without any fusion tags, and the integrity of this expression plasmid was confirmed by sequencing. The plasmid was transformed into the T7 express BL21 E. coli strain (New England Biolabs, Ipswich, MA) for protein expression studies. One liter cultures containing 100 µg ampicillin/mL of

LB media were grown at 37 ºC to an OD600 nm of 0.5-0.8 when the temperature was lowered to 28 ºC and additional ampicillin (100 µg/mL) was added. Protein expression relied on leaky T7 promoter activity. After overnight growth (12-16 h), cells were harvested by centrifugation and stored at -80 ºC. NOV2 was purified in the same manner as ACO [127] by ammonium sulfate fractionation and gel filtration chromatography. Purified protein samples (≥ 95% pure as judged by SDS-

PAGE analysis) were flash frozen and stored at -80 ºC or placed on ice for immediate use.

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4.2.3 Enzymatic assays and rate determination for background oxygen exchange

ACO activity studies were conducted according to previously established methods [127]. NOV2 enzymatic activity was assayed with resveratrol as a substrate in a 20 mM HEPES-NaOH, pH = 7.0. NOV2 steady-state kinetics was assessed by monitoring the reduction of resveratrol absorbance at 304 nm over time using a Flexstation 3 plate reader device (Molecular Devices, Sunnyvale, CA).

Reactions were carried at 28 ºC in 200 µL of 10 mM Bis-Tris-HCl, pH = 7.0, containing 0.8 µg of purified NOV2 and resveratrol (Sigma-Aldrich, St. Louis, MO,

99% purity) at eight concentrations ranging from 62.5 to 0.49 µM separated by two-fold dilutions. Absorbance measurements were taken every 15 sec for a total of 10 min. Initial reaction velocities were determined from the linear portions of the reaction profiles. Absorbance were related to absolute concentrations by simultaneous absorbance recordings of resveratrol standards. For NOV2 oxygen labeling studies, resveratrol was delivered in DMSO to a HEPES-NaOH, pH = 7.0 buffer system containing purified NOV2 and the reaction was carried out at 28 °C with 500 rpm shaking in a Thermomixer (Eppendorf, Hauppauge, NY), as described in detail below. Following the incubation, products were directly extracted by ethyl acetate and analyzed by HPLC (see below). Product amounts were quantified by plotting peak areas of known quantities of standards. To determine the background oxygen exchange for ACO and NOV2 catalyzed reaction products, the exchange reactions were performed in H218O. For ACO reaction products, the reaction mixture consisted of 192 µL H218O (99 % 18O atom,

Sigma-Aldrich), 4 µL of 1 M HEPES-NaOH, pH = 7.0, 1 µL of 10% (w/v) Triton X-

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100 and 3 µL 1 µg/µL of freshly purified ACO. Then either authentic all-trans-retinal

(TRC, Toronto, Canada, > 95% purity) or HPLC-purified 8’-hydroxy-15’-

apocarotenal generated in-house by a large scale ACO-catalyzed reaction, were

added to the reaction mixture. The reaction mixture then was incubated at 28 °C

with 500 rpm shaking in a thermomixer. Samples (20 µL) were removed from the

mixture at 0, 1, 2, 5, 15, 20 and 30 min and placed into tubes containing 200 µL

hexane followed by vigorous shaking. Then the extracted mixtures were

centrifuged in a bench-top centrifuge at 15,000 rpm for 2 min. Organic phases were

collected and analyzed by LC-MS (see below). Two additional experiments were

conducted in the same manner but with 1 % (w/v) BSA and/or 5 % (v/v) glacial

acetic acid to evaluate protein or pH-facilitated oxygen exchange. Background

oxygen exchange for the NOV2 reaction products was determined in a similar

manner. Briefly, the reaction mixture contained 150 µL H218O (97 % 18O atom,

Sigma-Aldrich), 3 µL of 1 M HEPES-NaOH, pH = 7.0, and 2.3 µL of freshly purified

NOV2 at 4.4 µg/µL. Authentic 4-hydroxybenzaldehyde (4-HBA) or 3,5-

dihydroxybenzaldehyde (3,5-DHBA) (Sigma-Aldrich, > 95% purity for both

compounds) dissolved in DMSO were added to the reaction mixture. Samples (20

µL) were removed from the mixture at 0, 2, 5, 10 and 15 min, placed into tubes

containing 200 µL of ethyl acetate, dried under argon, and then redissolved in

acetonitrile for MS analysis. As with the apocarotenoid product experiments

reactions containing 1 % (w/v) BSA or 5 % (v/v) glacial acetic acid were used as

controls.

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18 4.2.4 Isotope labeling study in H2 O

For the labeling study in H218O, cleavage reactions were performed the in same manner as described above. Specifically, 3 µL purified ACO at 1 µg/µL was added to a reaction mixture consisting of 192 µL H218O (99 % 18O atom, Sigma-

Aldrich), 4 µL of 1 M HEPES-NaOH, pH = 7.0, and 1 µL of 10% (w/v) Triton X-100.

The cleavage reaction was initiated by adding 5 µL of 4 mM all-trans-8’- apocarotenol dissolved in ethanol. The reaction mixture was incubated in a thermomixer at 28 °C with 500 rpm shaking for 5 min. Then the reaction was quenched with 200 µL methanol and 400 µL of diethyl ether/hexane (4:1, v/v) was added to extract the cleavage products. For the NOV2-catalyzed reaction, 2 µL of

25 mM resveratrol in DMSO was added to a 200 µL reaction mixture consisting of

194 µL H218O (97 % 18O atom, Sigma-Aldrich), 4 µL of 1 M HEPES-NaOH, pH =

7.0, and 2.3 µL of 4.4 µg/ µL purified NOV2 and this reaction was allowed to proceed for 5 min at 28 °C with 500 rpm shaking. The 4-HBA and 3,5-DHBA products of the reaction were then extracted with 500 µL ethyl acetate and immediately purified by normal-phase HPLC as described below. Peaks corresponding to each cleavage product were collected in glass tubes, dried under argon, and dissolved in acetonitrile. Both NOV2 products were separated by HPLC

(see below). ACO and NOV2 reaction products were further analyzed by mass spectrometry (MS) as outlined below.

18 4.2.5 Sample deoxygenation and isotope labeling study in O2

Sample deoxygenation was performed in 3 mL screw-capped glass vials with a gas-tight Teflon septum. A vial containing 1 mL of 20 mM HEPES-NaOH,

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pH = 7.0, was flushed with ultrapure argon (Airgas, Cleveland, OH) with constant

stirring. This deoxygenation treatment was carried out for different time to evaluate

the degassing efficiency. Oxygen supplementation was accomplished by flushing

the argon-purged buffer solution with O2 (Airgas) for 3 min. To test ACO activity in argon-purged or oxygen-supplemented buffer, 5 µL of Triton X-100 (10% w/v) and

7.5 µL of freshly purified ACO at 2 µg/µL was injected into the treated reaction

solutions with an airtight syringe (Hamilton, Reno, NV). The reaction was initiated

by injecting 10 µL of 4 mM all-trans-8’-apocarotenol in ethanol and allowed to

proceed for 3 min at room temperature with 800 rpm shaking. Then 1.5 mL of

methanol was injected to quench the reaction followed by addition of 1 mL diethyl

ether/hexane (4:1, v/v) to extract the products. The organic phase was collected

for LC/MS analysis. For the cleavage reaction performed in an 18O2 atmosphere,

30 µg of purified ACO and 25 µL of substrate at 4 mM in ethanol were used and

18O2 was introduced into the argon-purged reaction vial. The reaction was carried

out for 10 min, followed by product extraction and LC-MS analysis. Both the

deoxygenation and NOV2-catalyzed reaction in the presence of 18O2 were

conducted in the same way as for ACO. Following a 15 min argon purge of the

reaction solution, 5 µL of 20 mg/mL NOV2 and 10 µL of 25 mM resveratrol in

DMSO were injected to initiate the reaction, which was carried out for 3 min under

an 18O2 atmosphere. Reaction products were extracted with 500 µL ethyl acetate

and immediately dried by argon gas to remove the organic solvent and co-

extracted water. The dried sample then was redissolved in ethyl acetate, purified

by HPLC and further analyzed by MS.

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4.2.6 HPLC and mass spectrometric analyses of cleavage products

HPLC-MS analyses of the ACO-catalyzed cleavage products of all-trans-8’- apocarotenol were performed according to previously established methods [127].

Products of NOV2-catalyzed resveratrol cleavage were extracted with ethyl acetate, dried under argon, redissolved in ethyl acetate, and injected onto a normal phase Zorbax Sil column (5 µm, 4.6 × 250 mm) (Agilent, Santa Clara, CA).

Separation of the reaction products was achieved with hexane/ethyl acetate (3:2, v/v) as the mobile phase at a flow rate of 1.4 mL/min. Peaks eluted at ~4.2 min (4-

HBA) and ~5.3 min (3,5-DHBA) were individually collected, dried under argon, and dissolved in acetonitrile. Purified products from the isotope labeling experiments were directly injected into the APCI source of an LXQ linear ion trap mass spectrometer (Thermo Scientific, Waltham, MA) with acetonitrile and 10 mM ammonium formate. MS data were analyzed with the Xcalibur 2.0.7 software package. Incorporation of 18O was quantified by comparing relative ion intensities within the isotopic distribution window.

4.2.7 RPE microsome deoxygenation and isomerization activity assays

Bovine RPE microsomal membranes enriched in RPE65 were prepared as previously described [51]. The concentration of RPE65 in microsomal preparations was determined by quantitative densitometry on SDS-PAGE gels. Deoxygenation of RPE microsomes was carried out in the same manner as for ACO and NOV2.

Briefly, 0.5 mL of RPE microsomes in a 3 mL screw-capped glass vial with a gas- tight Teflon septum was purged with ultrapure argon. Afterwards, 2.5 µL of 20 µM all-trans-retinol in DMF was injected into the vial and the reaction and product

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analyses were performed as previously described [51]. O2 supplementation was accomplished by flushing the sample with O2 gas for 3 min before adding the substrate. The efficiency of O2 depletion/supplementation of RPE microsomes was assessed by monitoring the progress of ACO-catalyzed all-trans-8’-apocarotenol cleavage in the microsomal reaction mixture. Specifically, 7.5 µL of purified ACO at a concentration of 1 mg/mL were injected into the RPE65 reaction mixture after

O2 depletion/supplementation followed by injection of the apocarotenoid substrate.

Triton X-100 was omitted from those control experiments as it interferes with

RPE65 activity. The reaction and analysis were carried out as indicated above.

4.2.8 Protein crystallization, structural determination and analysis

Crystallization of T136A-ACO was conducted as previously described for the wild-type protein [127]. Briefly, 1.5 µL of purified enzyme at 10 mg/mL in 20 mM HEPES-NaOH, pH = 7.0 containing 0.02 % (w/v) Triton X-100 was mixed with a reservoir cocktail containing 0.1 M BTP-HCl, pH = 6.0, 21 – 23 % (w/v) sodium polyacrylate 2100 and 0.2 M NaCl in a 1:1 ratio. Crystallization was carried out by the hanging-drop vapor-diffusion method at 8 ˚C. Rod-shaped crystals typically appeared within 2 weeks. Mature crystals were directly harvested and flash cooled in liquid nitrogen before x-ray exposure. Diffraction data were collected at the NE-

CAT 24-ID-E beamline of the Advanced Proton Source (APS). The diffraction data were indexed, integrated and scaled with the XDS package [89]. Mutant T136A-

ACO crystals were isomorphous to previously reported orthorhombic wild-type

ACO crystals and their structures were determined by rigid body refinement in

REFMAC5 [92] using PDB entry 4OU9 as the starting model. Manual adjustments

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to the structure were made with COOT [172], and restrained refinement was

carried out in REFMAC5. Structures were validated using MOLPROBITY [95] and

the wwPDB structure validation server [135]. A summary of the X-ray data and

refinement statistics is shown in Table 4.1. All structural figures were prepared with

PyMOL (Schrödinger, New York, NY).

4.3 Results

4.3.1 Assessment of apocarotenoid solvent back-exchange

ACO-catalyzed cleavage of 8’-apocarotenol generates two aldehyde

products, all-trans-retinal (RAL) and a second C10-apocarotenal molecule (Fig.

4.1B). To quantify the solvent exchange rates of both aldehyde groups, RAL and

the C10-apocarotenoid were incubated in a 200 µL ACO reaction mixture

containing 3 µg of ACO with 18O-water (99% 18O atom) used as the reaction solvent.

The incubation was quenched and the apocarotenoids were extracted with hexane at different time intervals (0 – 30 min). The exchange rate was determined by

quantifying the ratio of target compound and its 18O-labeled counterpart that was

generated by the oxygen exchange. Virtually no detectable exchange was

observed within the time course of this experiment as observed previously [173]

(Fig. 4.2, A and B). However, the exchange rates for both products were

significantly increased by supplementing the reaction mixture with 1% (w/v) BSA

or by lowering the pH by adding 5% (v/v) acetic acid, both of which can catalyze

the oxygen exchange reaction (Fig. 4.2, A and B). For both apocarotenoids, about

80% of the carbonyl 16O atoms were substituted by 18O after 30 min incubation in an acidic environment with 1 % (w/v) BSA. Therefore, our data indicated that

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solvent back-exchange in our experimental conditions would not be a significant

complicating factor in the interpretation of the ACO labeling experiments.

18 4.3.2 Apocarotenoid labeling studies in the presence of H2 O

To assess the origin of the carbonyl oxygen atoms in the nascent

apocarotenoid products of the ACO reaction, activity assays were performed in the

presence of H218O/16O2, H216O/18O2 or H216O/16O2, the latter serving as a control.

In all experiments two peaks corresponding to RAL and the C10-apocarotenal

product were detected and identified based on their optical absorbance spectra

(Fig. 4.3A). The pseudomolecular masses [M + H]+ of m/z equal to 285 and 167

for products generated in H216O/16O2 were assigned to protonated RAL and C10

products, respectively (Fig. 4.2, C and D). For reactions carried out in an

H218O/16O2 environment, identical masses were observed for both products indicating that oxygen atoms in the RAL and C10-apocarotenal aldehyde groups originated from O2 rather than water (Fig. 4.2, E and F). As with the background

exchange experiments performed in the presence of BSA, high concentrations of

the enzyme might facilitate carbonyl oxygen solvent exchange. To test this

possibility, reactions were carried out in H218O using 1 mg of ACO (0.5 % w/v

protein). Here both products showed a remarkable increase in their m/z + 2

counterparts, suggesting that 16O atoms in the aldehyde products were replaced

by 18O from water (Table 4.2). These data strongly suggest that oxygen atoms in

the RAL and C10-apocarotenal aldehyde groups originated from O2 rather than water.

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18 4.3.3 Apocarotenoid labeling studies in the presence of O2

To further confirm the origin of the aldehyde oxygen atoms, additional

labeling studies were carried out in an 18O2 enriched environment. An efficient

method for removal of atmospheric O2 in the reaction solutions was first developed.

This procedure efficiently displaced dissolved O2 in the reaction mixture within 10

– 15 min, as shown by a drastic reduction in ACO-catalyzed RAL formation (Fig.

4.3B). Importantly, this RAL reduction could be restored by introducing O2 back

into the solution (Fig. 4.3B). In fact, O2 supplementation caused a ~2.5-fold

enhancement in ACO activity attributable to the higher concentration of dissolved

O2 in the reaction solutions.

Labeling studies in an 18O2-enriched environment were performed by

forcing 18O2 back into the reaction mixture following the deoxygenation treatment.

In this case, the isotopic distributions of both reaction products were shifted by 2

Da (Fig. 4.2G and H), indicating the formation of 18O-labeled products. Only small

amounts of 16O-labeled RAL and C10-apocarotenal (< 10%) were detected under

these conditions, likely generated by residual 16O-oxygen in the reaction mixture

and low levels of background solvent exchange (Table 4.2). Taken together, these

labeling studies in 18O-water and 18O2 environments unequivocally demonstrate

that ACO is a dioxygenase.

4.3.4 Preparation of highly purified and active NOV2

NOV2 is a stilbene oxygenase member of the CCO family that, amongst

other substrates, cleaves resveratrol to form 4-HBA and 3,5-DHBA (Fig. 4.1B).

Previously this CCO was characterized as a monooxygenase, indicating the

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possibility that different groups of CCOs could adopt different oxygenation mechanisms [123]. Thus, examination of the catalytic properties of NOV2 activity could reveal the determinants of mono- vs. dioxygenase activity in CCOs. The previous oxygen labeling study employed crude cell lysates from NOV2- expressing E. coli due to a substantial loss of activity after NOV2 was subjected to purification procedures. Excess E. coli protein in these labeling experiments could have significantly promoted solvent back-exchange in the aldehyde products, which would obfuscate the mono- vs dioxygenase assignment. To circumvent this limitation, we developed an expression and purification method for NOV2, based upon the protocol used for ACO that enabled the production of a highly pure and active protein sample (Fig. 4.4A and B). NOV2 expressed under reduced temperatures via leaky T7 promoter activity accumulated to very high levels in the cytosolic fraction of E. coli. A monodisperse and > 95% pure preparation of NOV2 was obtained by a simple purification scheme consisting of ammonium sulfate fractionation and gel filtration chromatography (Fig. 4.4A and B). With this homogeneous enzyme sample we carried out a steady-state kinetic study of NOV2 that revealed a kcat value similar to that of ACO [127, 174] (Fig. 4.4C). Notably,

NOV2 purified in this manner retained its activity for several weeks when stored on ice or at -80 ºC.

4.3.5 Assessment of the solvent back-exchange rate for 4-HBA and 3,5-DHBA

To measure solvent back-exchange in the benzaldehyde products of

NOV2-catalyzed resveratrol cleavage, authentic 4-HBA and 3,5-DHBA were incubated in a 150 µL H218O reaction mixture in the presence of 10 µg purified

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NOV2 for different periods of time. The aldehyde oxygen of 4-HBA remained

unchanged after 15 min incubation (Fig. 4.5A). By contrast, the carbonyl oxygen

in 3,5-DHBA was rather susceptible to solvent exchange with ~20% of oxygen

replaced by water-derived 18O during a 2 min incubation (Fig. 4.5B). Addition of 1%

(w/v) BSA into the reaction mixture significantly promoted the exchange for 3,5-

DHBA but not for 4-HBA. Lowering the pH also drastically accelerated the oxygen

substitution for both products with nearly 80% of the original oxygen atoms

replaced by those from water (Fig. 4.5A and B). These results suggested that the

oxygen back-exchange would not significantly cloud the interpretation of product

oxygen labeling in the NOV2-catalyzed reaction.

18 4.3.6 NOV2 labeling studies in the presence of H2 O

The NOV2-catalyzed resveratrol cleavage reaction was performed in

H216O/16O2 and H218O/16O2 and H216O/18O2. HPLC analysis of the products obtained in H216O/16O2 showed two peaks corresponding to 4-HBA and 3,5-DHBA.

The identity of each product was confirmed by their absorbance spectra (Fig. 4.6A).

Subsequent MS analysis of the products from H216O/16O2 showed that the

pseudomolecular masses of m/z of 121 and 137 could be assigned to

deprotonated 4-HBA and 3,5-DHBA ([M - H]-), respectively (Fig. 4.5C and D). The product isotopic distribution patterns were similar when the reaction was performed in H218O/16O2 (97% 18O) (Fig 5E and F compared to C and D) with a slightly higher

content of the m/z = 139 species (~10%) for 3,5-DHBA that was attributable to

solvent back-exchange. These labeling results suggested that the carbonyl oxygen

atoms in the final products originated from molecular oxygen rather than water.

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18 4.3.7 NOV2 labeling studies in the presence of O2

18O2 labeling experiments for NOV2 were carried out in deoxygenated buffer in the same manner as described above for ACO. Removal of atmospheric O2

resulted in a dramatic reduction in 3,5-DHBA production by NOV2 that could be

restored by reintroduction of O2 gas (Fig. 4.6B). MS analysis of products generated

in 18O2 atmosphere during the 1 min reaction revealed a 2 Dalton shift for 4-HBA molecule compared to the mass observed from reactions carried out in standard

O2 (Fig. 4.5G). Similarly, the isotopic distribution patterns revealed 78.1% of 3,5-

DHBA molecules become 18O-labeled (Fig. 4.5H). Because 3,5-DHBA has a

readily exchangeable aldehyde oxygen, it is likely that this unlabeled 3,5-DHBA

species (21.9%) may be generated by solvent back exchange. Extension of the

assay incubation time to 2 min only slightly reduced the percentage of 3,5-DHBA

carrying an 18O label (Table 4.3). However, when the sample was dried in a

SpeedVac under heat (~15 min) rather than under a stream of argon (~2 min), the

unlabeled 3,5-DHBA species substantially increased to 35% (Table 4.3), likely due to the elevated temperature and increased time of exposure to water. Therefore, this indicated that seemingly minor differences in sample preparation can have a significant impact on the experimental outcome. Taken together, the high incorporation rate of 18O from H216O/18O2 and 16O from H218O/16O2 into both

products demonstrated that NOV2 is a dioxygenase.

4.3.8 Activity and structure of T136A-ACO

A density functional theory (DFT) study of the ACO reaction mechanism

found that Thr136 could promote a dioxygenase type mechanism by limiting

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access of water to the iron center [34]. The non-heme iron center of wild-type ACO adopts an octahedral-like structure with four coordination sites occupied by His ligands and a fifth, trans to His183, occupied by solvent. The sixth site is occluded by the methyl group of Thr136, positioned ~4.6 Å away from the Fe(II) atom. This arrangement creates a hydrophobic microenvironment around the sixth coordination site that likely impedes its accessibility to water [12]. DFT calculations indicated that the coordinated solvent is displaced by O2 during catalysis, which could potentially bind a side-on or end-on fashion. A structure-based sequence alignment revealed that most CCOs contain a hydrophobic or semi-hydrophobic residue (Thr, Val or Ile) at this position (Fig. 4.7A and B). A reduction in the bulkiness at this site could create enough space for water to coordinate iron and allow it to participate in the reaction. In fact, VP14, one of the few CCOs possessing a non-bulky Ala residue at this position (Fig. 4.7A) was demonstrated to bind water at this site [175]. If the ACO reaction occurs through an epoxide intermediate, an iron-coordinated solvent molecule could act as a nucleophile to open the epoxide, which would give rise to a monooxygenase labeling pattern.

To test these hypotheses, we generated a T136A version of ACO, in which the sixth coordination site was expected to be water accessible and examined its catalytic activity and labeling pattern. Cleavage of 8’-apocarotenol by this mutant enzyme was severely impaired with minimal product formation observed using the standard reaction protocol. The poor activity could nevertheless be overcome by increasing the amount of enzyme used in the assay. In reactions containing 100

µg of T136A-ACO, RAL formation after a 10 min incubation was approximately 50%

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of that formed by wild-type protein over the same period of time (Fig. 4.8A). Using

these modified conditions, we carried out a labeling study using T136A-ACO in the

presence of H218O. We observed that the RAL and C10-apocarotenal products

generated by T136A-ACO were labeled primarily with 16O similar to those generated by the wild-type enzyme (Fig. 4.8B and C compared to D and E). In both

cases, the reaction products showed an increased extent of 18O labeling compared

to those produced under standard assay conditions by wild-type protein. This

elevation is attributable to an increase in non-specific protein-catalyzed oxygen

exchange caused by the large quantities of enzyme used in the experiments.

A crystal structure of T136A ACO confirmed that this substitution could

theoretically allow for the binding of water to the coordination site trans to His 304.

The ~6 Å gap separating the Ala136 methyl side chain and the iron center could

easily accommodate an iron-bound solvent molecule with minimal steric hindrance

(Fig. 4.7C). However, rather than 6-coordinate octahedral geometry with water

molecules occupying the vacant sites, we instead observed a 5-coordinate trigonal

bipyramidal structure with a single water molecule bound to the iron. Additionally,

there was a slight but consistent increase (average 0.26 Å) in the Fe-His bond

lengths compared to the wild-type protein (Fig. 4.7B and C). Together, these data

demonstrated that the Thr136 side chain is not a factor dictating the dioxygenase

labeling pattern of ACO; however, it does appear to be critical for overall catalytic

activity and structural integrity of the iron center.

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4.3.9 Influence of O2 on RPE65 retinoid isomerase activity

RPE65 is an atypical CCO that catalyzes ester cleavage and isomerization of all-trans-retinyl esters instead of oxidative cleavage of carotenoid substrates [86]

(Fig. 4.1B). Although not necessarily expected based on the chemistry being performed, the question of whether O2 is required for this important reaction has not been experimentally evaluated. Exploration of this possibility is certainly warranted given the close phyletic relationship of RPE65 to the oxygen-utilizing

BCO1 and BCO2 enzymes (Fig. 4.1A), as well as the presumed structural similarity of their iron centers. To address this question, we compared RPE65 isomerase activity in native RPE microsomes before and after sample deoxygenation. To evaluate the extent of O2 removal from the microsome-containing sample we measured, in parallel, the apocarotenoid oxygenase activity of ACO that was exogenously added to the RPE65 reaction mixture, serving as an internal control.

Importantly, ACO activity in the RPE65 reaction system was comparable to that obtained in standard reaction conditions (Fig. 4.9A). Similar to what was observed under standard conditions (Fig. 4.3B), ACO activity was markedly impaired following argon treatment to remove O2 with a 3.4-fold reduction in turnover number. The activity was fully rescued and in fact augmented by reintroduction of

O2 into the reaction system (Fig. 4.9B). ACO thus served as a reliable indicator of the oxygen concentration in the RPE65 reaction system. When RPE65 activity was measured following the same deoxygenation treatment the amount of formed

11cROL was only marginally decreased (~14%), and the reduced activity could not be restored by reintroduction of O2 into the reaction mixture (Fig. 4.9B).

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Moreover, O2 supplementation of the reaction mixture without prior deoxygenation

actually depressed the activity by ~30% in contrast to the ~34% activity boost seen

for ACO (Fig. 4.9B). This loss of RPE65 activity in the presence of excess O2 could

be caused by oxidation of the iron cofactor which is required to be its ferrous form

to be catalytically competent [33]. Importantly, the total number of substrate turnover events was 3.6 times higher for RPE65 compared to ACO, which rules

out the possibility that a reduced O2 requirement by RPE65, due to less overall catalytic activity, could explain its insensitivity to O2 concentration (Table 4.4).

Collectively, these results support an O2-independent mechanism for RPE65-

catalyzed retinoid isomerization.

4.4 Discussion

The question of whether CCOs are monooxygenases or dioxygenases,

although seemly straightforward to determine experimentally, has remained

contentious despite several published studies addressing the subject. Major

difficulties and problems associated with these studies include their use of crude

cell extracts as a source of enzymatic activity, the low activity of CCO enzymes in

general, inclusion of only one of the two cleavage reaction products for analyses

of isotopic labeling and, most importantly, solvent back-exchange of the aldehyde

cleavage products facilitated by both high protein concentrations and long

incubation times. Taken at face value, discrepancies between these studies might

also indicate that both monooxygenases and dioxygenases could exist within the

CCO family. With these issues in mind, we set out to examine the oxygen labeling

pattern of the well-characterized, prototypical CCO, Synechocystis ACO as well as

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a distantly related, stilbene-cleaving bacterial CCO called NOV2 that was previously classified as a monooxygenase.

Capitalizing on our recent success in generating a highly purified, native

ACO with robust and durable enzymatic activity, we found that this enzyme exhibits

a dioxygenase pattern of O2 incorporation into its reaction products. This result is

consistent with a previous computational study on the ACO reaction mechanism,

which favored a dioxetane-based mechanism that would cause both oxygen atoms

of O2 to appear in the reaction products [34]. A key prediction from this theoretical

study was that the ability of a vacant site in the iron coordination sphere to bind

water could govern whether the reaction occurs through a dioxetane or an epoxide

intermediate, which could give rise to either dioxygenase or monooxygenase

labeling patterns in the reaction products, respectively. This site, located trans to

His304, is blocked in ACO by the hydrophobic methyl group of Thr136. We

replaced Ala for Thr at this position to potentially remove the steric and electrostatic

barriers to solvent binding at the sixth coordination site. Notably, VP14 has a

naturally occurring Ala residue at the corresponding position in its sequence and

the reported crystal structure contains bound solvent at the coordination site in

question [175]. Despite a major reduction in catalytic activity caused by the T136A

substitution, the dioxygenase labeling pattern for the enzyme was maintained. The

crystal structure of T136A-ACO showed that the iron center remained five-

coordinate just like the native enzyme even though the coordination geometry

adopted a trigonal bipyramidal structure due to a shift in the coordinated solvent

molecule. Thus, the dioxygenase activity of ACO is resistant to changes in solvent

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coordination potential of its iron center. Given the reported ability of the VP14 iron

center to accept water in its sixth coordination site, it would be of interest to

examine the oxygen labeling pattern of this enzyme which, despite some claims to

the contrary, has not yet been properly investigated.

Our labeling experiments with the stilbene-cleaving CCO, NOV2, previously

described as a monooxygenase [123], clearly demonstrated a dioxygenase pattern

of oxygen incorporation into its benzaldehyde reaction products. What are the

causes for this discrepancy in experimental outcomes? Quantification of oxygen

labeling in the prior study indicated a substantial amount of both 4-HBA (69%) and

3,5-DHBA (35%) were labeled with 18O when the reaction was performed in an

18O2/H216O environment. Such a labeling pattern could be obtained through a monooxygenase-generated epoxide intermediate that is opened in a non- regioselective manner to yield a mixture of 16O and 18O in each product. Importantly,

because the samples were not deoxygenated prior to initiation of the reaction, a

significant amount of product could have been labeled with atmospheric 16O2 thus

lowering the incorporation of 18O2 into the products. However, this explanation for

the labeling results is in conflict with the labeling pattern observed in the same

study from reactions performed in H218O/16O2 in which very little (~10%) 4-HBA was labeled with 18O. These prior experiments employed crude NOV2-containing

cell lysate as an enzyme source, and the contaminating proteins could have

exacerbated solvent back-exchange. The reactions in this study were also carried

out for 15 min, a period of time that, under our reaction conditions, allows

substantial solvent back-exchange to occur for 3,5-DHBA. Thus it appears likely

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that the prior analyses were confounded by unappreciated solvent back-exchange.

This difficulty was circumvented in the present experiments by the use of highly purified and active NOV2, which enabled the use of low protein concentrations and short reaction times. Back-exchange was further minimized by employing water- free normal-phase HPLC conditions for product purification and MS analyses together with rapid solvent removal from the product-containing extracts.

Our understanding of carotenoid cleavage by CCOs would greatly benefit from a genuine high-resolution CCO-substrate structure, which has yet to be reported. A major difficulty in obtaining such a complex relates to the extreme hydrophobicity of carotenoid substrates, which limits their aqueous solubility and prevents stoichiometric formation of ES complexes for structural studies. On the other hand, hydroxylated stilbene substrates of the lignostilbene-cleaving CCOs, such as resveratrol, exhibit much greater water solubility and could be more amenable to structural studies. Our demonstration that NOV2 is a dioxygenase indicates that this enzyme may serve as a reliable model system for studying the general mechanism of alkene cleavage by CCOs. We have described a straightforward expression and purification procedure that results in ample quantities of purified and active NOV2 for structural and spectroscopic studies of the alkene cleavage reaction.

Taken together with prior studies, our demonstration of dioxygenase activity in a primitive cyanobacterial carotenoid-cleaving CCO, as well as the lignostilbene- cleaving CCO, NOV2, strongly suggest that all alkene-cleaving CCOs are dioxygenases that effect the double bond cleavage reaction by a common catalytic

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mechanism. It should be noted that a dioxygenase or monooxygenase labeling

pattern does not necessarily imply a specific mechanism of O2 incorporation into the substrate. For example, DFT calculations on carotenoid cleavage by ACO have shown that a dioxygenase labeling pattern could be achieved through either dioxetane or epoxide reaction intermediates [34]. Future biophysical studies are needed to delineate the precise mode of catalysis by alkene-cleaving CCOs.

In contrast to other CCOs, we have shown here that RPE65 does not rely on O2 to catalyze ester cleavage/isomerization of all-trans-retinyl esters. This result

is not unexpected given that transformation of all-trans-retinyl esters into 11-cis-

retinol does not entail any redox chemistry. Moreover, the hydroxyl oxygen atom

in the 11-cis-retinol product has been directly shown to originate from water [14].

However, it has recently been proposed that O2 may play a catalytic role in

retinoid/carotenoid isomerization by bonding with C12 of the retinoid backbone to

generate a temporary C11-C12 single bond that would allow free bond rotation to a

cis-like state followed by oxygen dissociation to restore the polyene conjugation in

an 11-cis configuration [166]. Even though O2 does not stoichiometrically participate in this proposed reaction, a major reduction in O2 concentration, as

accomplished by our deoxygenation procedure, should still greatly slow the rate of

retinoid isomerization, which is not what we observed. The lack of activity reduction

after deoxygenation also cannot be explained by potential tight binding of O2 to the iron center as our prior structural and spectroscopic studies of RPE65 did not reveal such a stable Fe-O2 complex [14, 176]. Most importantly, a recent structural

determination of RPE65 in complex with a retinoid mimetic indicates that the

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polyene chain of carotenoids binds at a position distant from the iron center, which excludes formation of an Fe-O2-retinoid complex [167]. Thus the commonality in reaction mechanisms between alkene-cleaving CCOs and RPE65 appears unrelated to their O2 requirements but rather to their use of iron to bind oxygen: for

O2 activation in the former and as an ester-polarizing Lewis acid in the latter.

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Tables

Table 4.1. X-ray crystallographic data collection and refinement statistics for

T136A-ACO

Data collection a

Beamline NECAT 24-ID-E

Wavelength (Å) 0.9793

Space group P212121

Unit cell parameters (Å) a = 118.76

b = 125.96

c = 203.91

Resolution (Å) 48.61-2.8 (2.90-2.80)a

Unique reflections 75,385 (11,999)

Completeness (%) 99.81 (99.05)

Multiplicity 3.73 (3.77)

7.07 (0.73)

RmeasI (%)b 25.1 (219.6)

CC1/2 (%)b 98(18.8)

Refinement

Resolution (Å) 48.6-2.8

No observations 71479

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Rw ork/Rfree (%)c 22.5/26.4

No atoms

Protein 15,090

Water 44

Metal/ion 4 Fe, 2 Cl

B-factors (Å2)

Protein 63

Water 38

Metal/ion 49(Fe), 56(Cl)

RMS deviations

Bond lengths (Å) 0.010

Bond angles (°) 1.40

Ramachandran plotd

Favored/outliers (%) 96/0

PDB accession code 5E47 a Values in parentheses are those for the highest resolution shell of data b As calculated in XDS c As calculated in REFMAC d As accessed with MolProbity

157

Table 4.2. Summary of isotope labeling results for ACO.

16O2-H218O system 18O2-H216O system

Compound

16O-labeled 18O-labeled 16O- 18O-

labeled labeled

all-trans-retinal 97.7 % 2.2 % 7.8 % 92.2 %

(71.8 %)* (28.2 %)

C10- 97.9 % (89.4 %) 2.1 % 6.8 % 93.2 %

apocarotenal (10.6 %)

* Numbers in parentheses are the results obtained in a reaction system containing 1 mg of ACO

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Table 4.3. Summary of isotope labeling results for NOV2.

Compound 16O2-H218O system 18O2-H216O system

16O-labeled 18O-labeled 16O-labeled 18O-labeled

4-HBA 98.7 % 1.3 % 4.5 % 95.5 %

3,5-DHBA 92.2 % 7.8 % 21.9 % 78.1 %

(24.1 %)a (75.9 %)a

(35.1 %)b (65.9 %)b a Reaction products dried by argon stream after a 2 min reaction b Reaction products dried by SpeedVac method after a 2 min reaction

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Table 4.4. Enzymatic turnover number of ACO and RPE65 before and after deoxygenation treatment

Enzyme kcat kcat (Ar Total number of turnovers per

(untreated) treated) molecule (untreated reactions)

ACO 1.09 ± 0.13 0.32 ± 0.03 5.5

RPE65 0.33 ± 0.01 0.28 ± 0.02 20

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Figures

Figure 4.1. Phylogenic and enzymatic relationships amongst CCOs. A, An unrooted maximum likelihood phylogenic tree of selected CCO family members.

The tree termini are colored according the substrate specificity of the associated taxon/clade. The BCO1, BCO2 and RPE65 clades are composed of sequences from Mus musculus, Rattus norvegicus, Homo sapiens and Bos taurus. The altitudes of the triangles associated with these clades are proportional to the evolutionary diversity amongst the examined taxa. Bootstrap values from 1000 pseudo-replicates are displayed as percentages beside the associated branches.

The scale bar indicates the average number of site substitutions over the indicated distance. Members of the CCO family whose reaction mechanisms have previously been examined by isotope labeling experiments are shown in bold.

Abbreviations are as follows: Drosophila melanogaster NinaB, dNinaB; Galleria mellonella NinaB, gNinaB; Synechocystis sp. PCC 6803 ACO, ACO; Nostoc sp.

PCC 7120 carotenoid oxygenase, NSC; Novosphingobium aromaticivorans CCO

2, NOV2; Pseudomonas nitroreducens isoeugenol monoxygenase, IEM; Ustilago maydis resveratrol cleavage oxygenase I, umRCO I; Neurospora crassa carotenoid oxygenase I, CAO I; Pseudomonas paucimobilis lignostilbene dioxygenase I, LSD I; U. maydis β-carotene cleavage oxygenase I, umBCO1;

Fusarium fujikuroi carotenoid oxygenase, fCARX; Zea mays viviparous 14, VP14;

Arabidopsis thaliana carotenoid cleavage dioxygenase 1, atCCD1. B, Reactions catalyzed by CCOs considered in the present study. Synechocystis ACO, a prototypical CCO member, specifically cleaves all-trans-8’-apocarotenol at the

161

15,15’ double bond position to generate all-trans-retinal (RAL) and 8’-hydroxy-15’- apocarotenal (C10-apocarotenal). RPE65, an atypical CCO member, catalyzes a coupled ester cleavage/isomerization of all-trans-retinyl esters (atRE) rather than oxidative carotenoid cleavage. This reaction produces 11-cis-retinol (11cROL), a key intermediate in the regeneration of visual chromophore required for vertebrate vision. NOV2 from Novosphingobium cleaves resveratrol at the interphenyl double bond to produce 3,5-dihydroxybenzaldehyde (3,5-DHBA) and 4- hydroxybenzaldehyde (4-HBA). Wavy red lines indicate the location of the scissile bond in each reaction.

162

Figure 4.1.

163

Figure 4.2. In vitro isotope-labeling analysis of the ACO-catalyzed reaction.

Assessment of solvent back-exchange rate for A, RAL and B, C10-apocarotenal.

Authentic RAL or C10-apocarotenal were added to a buffered reaction system

containing H218O (99% 18O) and purified ACO, and the mixture was incubated at

28 ºC with 500 rpm shaking. Samples were collected at the indicated time points.

Solvent back-exchange rates for RAL and C10-apocarotenal were quantified as

the ion peak area ratios of the 16O- and 18O-labeled species. Note the increased

exchange rates induced by addition of 1 % (w/v) BSA and/or 5 % (v/v) acetic acid.

Error bars represent S.D.s from duplicate measurements. Mass spectra for C, RAL

and D, C10-apocarotenal generated under standard H216O/16O2 conditions. E and

F, Mass spectra for the apocarotenoid products generated in an H218O/16O2

environment showed isotope distributions similar to those of the control spectra

shown in panels C and D, which demonstrated a lack of 18O incorporation from the labeled water into the products. G and H, By contrast, spectra for both

apocarotenoids generated in an H216O/18O2 milieu showed a 2 Da shift in their

isotope distribution patterns, attributable to 18O incorporation into both products.

The data are quantitated in Table 4.2.

164

Figure 4.2.

165

Figure 4.3. HPLC analysis of ACO-catalyzed reaction products and the

influence of O2 depletion and supplementation on ACO enzymatic activity. A,

HPLC trace of ACO-catalyzed reaction products generated from cleavage of all-

trans-8’-apocarotenol under an 18O2-gas environment. The peak assignments

were made based on their elution times and their characteristic absorbance

spectra as shown to the right of the HPLC chromatogram. B, ACO activity assays

were performed either without argon pretreatment to displace O2 (control) or after

a 10 or 15 min argon purge (white bars) and compared with ACO samples

undergoing the same treatments but subjected to 3 min of O2 gas supplementation as a final step (gray bars). Error bars represent S.D.s calculated from measurements performed in triplicate.

166

Figure 4.3.

167

Figure 4.4. NOV2 purification and enzymatic characterization. A, Gel filtration chromatogram showing a symmetrical peak (indicated by the arrow) for NOV2 obtained from ammonium sulfate fractionation. Comparison of the elution volume of this NOV2 peak with those of standards indicates NOV2 is a monomeric protein with a molecular weight of ~54 kDa. B, SDS-PAGE analysis of pooled gel filtration fractions constituting the main NOV2 peak. Proteins were visualized by Coomassie

R-250 staining. C, Steady-state kinetics of purified NOV2. Error bars represent standard deviations computed from triplicate measurements. Uncertainty estimates for the kinetic parameters are standard errors computed from the curve fitting algorithm in SigmaPlot.

168

Figure 4.4.

169

Figure 4.5. In vitro isotope-labeling analysis of the NOV2-catalyzed reaction.

Assessment of solvent back-exchange rates for A, 4-HBA and B, 3,5-DHBA.

Authentic 4-HBA and 3,5-DHBA were added to a buffered reaction system

containing H218O (97% 18O) and purified NOV2, and the mixture was incubated at

28 ˚C with 500 rpm shaking. Samples were collected at the indicated time points.

Solvent back-exchange rates for 4-HBA and 3,5-DHBA were quantified as the ion

peak area ratios of the 16O- and 18O-labeled species. Note the greater susceptibility of 3,5-DHBA to solvent back-exchange compared to that of 4-HBA, as well as the

increased exchange rate for both compounds in the presence of 1 % (w/v) BSA or

5 % (v/v) acetic acid. Error bars represent S.D.s from experiments performed in

duplicate. Mass spectra for C, 4-HBA and D, 3,5-DHBA generated under standard

H216O/16O2 conditions. E and F, Mass spectra for the cleavage products generated

in an H218O/16O2 environment showed isotope distributions similar to those of the

control spectra in panels C and D, which demonstrated a lack of 18O incorporation from the labeled water into the products. G and H, By contrast, spectra for the two

products generated in an H216O/18O2 environment showed a 2 Da shift in their isotope distribution patterns, attributable to 18O incorporation into both compounds.

A quantitative analysis of the data is presented in Table 4.3.

170

Figure 4.5.

171

Figure 4.6. HPLC analysis of NOV2-catalyzed reaction products and the

influence of O2 depletion and supplementation on NOV2 enzymatic activity.

A, HPLC trace of NOV2-catalyzed reaction products generated from cleavage of

resveratrol. The peak assignments were made based on their elution times, in

comparison to authentic standards, and their characteristic absorbance spectra as

shown to the right of the HPLC chromatogram. B, NOV2 activity assays were

performed either without argon pretreatment to displace O2 (control) or after a 10

or 15 min argon purge (white bars) and compared with NOV2 samples undergoing

the same treatments but subjected to 3 min of O2 gas supplementation as a final

step (gray bars). Error bars represent S.D.s calculated from measurements

performed in triplicate.

172

Figure 4.6.

173

Figure 4.7. Active site structure of wild-type and T136A ACO. A, A structure-

based sequence alignment of selected CCOs [177, 178]. The position in the

sequences homologous to Thr136 of Synechocystis ACO (Novosphingobium

NOV2, bovine RPE65, maize VP14 and human BCO1) is marked with a blue star.

A conserved Glu residue that participates in second shell iron coordination is

marked by a blue square. B, Structure of the wild-type ACO Fe(II)-center showing

a 5-coordinate partially-filled octahedral geometry. C, Structure of the T136A-ACO

Fe(II)-center which has a structure more consistent with trigonal bipyramidal

geometry owing to a change in the binding position of the coordinated solvent.

Notably, no extra electron density that would indicate the presence of a

coordinated solvent molecule between Ala136 and iron was observed. Blue mesh

represents 2mFo – DFc electron density contoured at 1 RMSD and computed without inclusion of the iron-bound solvent molecules in the structural models.

Bond lengths (in Å) between Fe(II) and nitrogen atoms that form the 4-His coordination shell are shown. Iron and the iron-bound water molecules are

depicted as orange and red spheres, respectively.

174

Figure 4.7.

175

Figure 4.8. Activity and in vitro isotope-labeling analysis of T136A-ACO. A,

Activity results of T136A and wild-type ACO. 100 µg of purified protein were used in each reaction. Enzyme activity was assessed by following the formation of RAL over time. Note that the substrate is completely consumed by the wild-type protein within the first minute of the reaction; therefore, product formation in this graph cannot be used to compare the activity levels of wild-type and T136A ACO. B and

C, Mass spectra of apocarotenoid products generated by wild-type ACO in an

H218O/16O2 environment. Note the increased levels of 18O-labeled RAL (m/z=287.2) and C10-apocarotenal (m/z=169.1) generated as a result of the high protein concentration used in the assay. D and E, Mass spectra of apocarotenoid products generated by T136A-ACO in an H218O/16O2 environment were highly similar to those in panels B and C, which indicated the preservation of dioxygenase activity in the ACO point mutant.

176

Figure 4.8.

177

Figure 4.9. Influence of O2 levels on the retinoid isomerase activity of RPE65.

A, ACO enzymatic activity, as monitored by the formation of RAL from 8’- apocarotenol, is maintained when the reaction is carried out in the RPE65 retinoid isomerization assay mixture. B, Influence of O2 depletion or supplementation on

ACO and RPE65 enzymatic activity. Whereas ACO activity is markedly affected by manipulations in O2 concentration within the reaction buffer, RPE65 activity is

indifferent to either O2 supplementation or depletion. Each group was either

supplemented for 3 min with O2, purged with argon for 1 h or supplemented with

O2 for 3 min following a 1 h argon purge. Error bars represent S.D.s calculated

from triplicate measurements.

178

Figure 4.9.

179

CHAPTER 5: SUMMARY AND FUTURE DIRECTIONS

180

5.1. Significance of the thesis project

Since the discovery of the first CCO gene, our understanding of the physiological functions associated with this growing family of enzymes has improved significantly. Recent biochemical and structural characterizations of selected CCOs have provided insights into their biochemical and catalytic properties. Despite encouraging progress, a detailed CCO catalytic mechanism is still lacking. The relatively slow progress in this area can be attributed to the experimental difficulties associated with CCOs, including i) their resistance to homo/heterologous expression typically resulting in low levels of expression or misfolded proteins; ii) the impurity of protein preparations and lack of enzymati c activity; iii) the absence of a convenient method for rapid enzymatic studies; and iv) difficulties in crystallization and structural characterizations.

We chose ACO as a model CCO for in depth study. First, ACO is a primitive member of the CCO family and, according to our initial tests, it was amenable to heterologous expression. Of further interest, previous structural characterizations of this enzyme revealed that ACO, in addition to its canonical carotenoid cleaving function, also possesses isomerase activity. Therefore, its combined cleaving and isomerizing activities make ACO an attractive CCO for further study. To overcome the difficulties noted above, we developed an efficient protein expression and purification method which yields large amounts of pure protein sample with robust activity. To better study the kinetic features of the enzyme, we also designed an efficient spectroscopic method. This significantly reduced the labor and time for

ACO kinetic studies. Overall, these experimental breakthroughs substantially

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facilitated our biochemical characterizations. Importantly, the expression method for ACO also proved successful for NOV2. Therefore, our expression methodology for bacterial CCO likely can be applied to other CCOs. Our subsequent biochemical and crystallographic characterizations of ACO and NOV2, as discussed below, shed important insights into the CCO catalytic mechanism.

5.1.1. Insights into the CCO isomerase hypothesis

The previous ACO crystallographic study by Kloer et al [12] suggested an unconventional isomerase activity associated with this prototypical CCO, and this proposal was further supported by biochemical evidence showing that trace amount of cis-configured retinal products were observed in ACO-catalyze d reactions. As isomerization activity was associated with a few other CCO members, including RPE65 and NinaB, results obtained from ACO implied that the trans-to- cis activity may be common to all CCOs. Consequently, this isomerase hypothesis gained considerable attention in the CCO field.

To further test the hypothesis, we studied the same CCO and our findings did not support the proposed isomerase activity. First, we provided biochemical evidence showing that the cis-product observed in previous studies was actually generated by photo- and/or thermo-isomerization. Our subsequent crystallographic characterizations of ACO demonstrated that the electron density signal in the active site observed in the previous study of Kloer et al., key evidence for the isomerase hypothesis, may simply represent a bound linear detergent used for crystallization rather than the substrate molecule. Subsequent in situ stop-flow assays by Raman spectroscopy further supported our conclusions about the

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isomerase activity. Therefore, our study has resolved the long-standing isomerase controversy in the field. Further, we propose that isomerase activity is associated with only a few CCOs (e.g., RPE65 and NinaB) with highly specialized biological functions (e.g., vision), and that most CCO family members only display oxygenase activity. More importantly, our results also demonstrated the influence of detergent on CCO activity and indicated that careful selection of the detergent is critical for the success of structural studies aimed at elucidating structures of

CCO-carotenoid complexes. Finally, our extensive but unsuccessful efforts to obtain the crystal structure of ACO complexed with substrate/product indicate that obtaining the complex structure is quite challenging due to the extreme hydrophobicity of carotenoids/apocarotenoids.

5.1.2. Structural basis for substrate selectivity and regiospecificity

As a common feature, CCOs often display a high degree of substrate promiscuity. However, they also exhibit high regio- and stereo-selectivity pertaining to the scissile double bond for cleaving and to the polyene isomeric configurations of the carotenoid substrates. Those distinct enzymology features likely are attributable to the structural and amino acid compositions defining the

CCO’s substrate binding tunnel. Despite progress towards more definitive CCO structural determinations, however, the biochemical and structural basis underlying these interesting enzymatic features are remain uncertain.

We began by systematically examining the functional and structural importance of ACO active site residues. The results of expressing proteins with active site mutations clearly demonstrated that the residues in the substrate

183

binding pocket have little significance for protein folding. The unaltered regiospecificity towards the C15-C15’ cleavage site by each mutant enzyme demonstrated that those residues within the substrate binding tunnel work in concert and construct a rigid substrate processing platform to guarantee regiospecificity. Subsequent kinetic studies of each mutant further confirmed the structural rigidity of the substrate binding region. Collectively, we obtained biochemical evidence demonstrating that CCOs possess a high degree of accuracy in substrate binding and processing which could not be altered easily by active site mutations.

5.1.3. Functions of the 3-Glu outer Fe-coordination sphere

Our biochemical and structural studies of ACO mutants of Glu150Asp and

Glu150Gln demonstrated the functional significance of the conserved 3-Glu iron- outer coordination sphere in CCOs. The non-charged 4-His iron binding motif in

CCOs appears incompetent for metal charge balancing and thus requires an extra ligand for iron binding. The negatively charged 3-Glu outer sphere could perfectly serve this role by elevating the pKa of the inner sphere imidazole rings, which in turn stabilize the His-Nε-iron coordinate bond and tune the reactivity of Fe(II) towards dioxygen. Our mutagenesis and crystallographic studies clearly demonstrate the role of the 3-Glu shell in iron charge balancing to guarantee stable iron binding in the catalytic center. Of further significance, the negative sidechain of the Glu150 residue may also facilitate the stabilization of the carotenoid carbon cation, a reaction intermediate that is believed to occur during CCO-catalyze d reactions. The importance of the conserved Glu residue observed in our studies

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may explain the molecular pathogenesis of severe juvenile blindness associated with the E148D RPE65 point mutation within the same region.

Interestingly, the negatively charged residues in the metal-outer binding sphere, which are important for enzyme function, have been observed in several other metalloenzymes, including 15-lipoxygenase. However, little is known about their function in enzyme catalysis. Considering the role of the 3-Glu sphere in

CCOs, it will be interesting to test their similar functions (i.e., charge-balancing) as in CCOs and/or other novel functions in other metalloenzymes.

5.1.4. Resolving the controversy of the CCO oxygenation mechanism

The long-standing debate about whether a mono- or dioxygenase mechanism is utilized by CCOs prompted us to investigate the oxidative pattern in

ACO. For a more reliable conclusion, the oxygen back-exchange rate for each reaction product was closely monitored in our study. In addition, the method developed in our 18O2 labeling experiment significantly reduces the background noise by regular dioxygen. With those improvements, our isotope labeling study of

ACO unequivocally demonstrated a dioxygenase pattern for the cleavage reaction.

In addition, our labeling experiments of NOV2, which was previously thought to act as a monooxygenase, indicated this stilbene-cleaving CCO, just like its distant relatives that recognize carotenoids, is utilizing a dioxygenase mechanism. The previous characterization of NOV2 as a monooxygenase could due to the enzyme’s low activity and therefore the relatively long incubation times required with a large amount of semi-purified enzyme. In our experiments, the highly purified NOV2 with robust activity clearly showed that this stilbene-cleaving

185

CCO adopts the same reaction mechanism as other CCOs. In combination with previous isotope-labeling studies of plant and mammalian CCOs, our results on

ACO and NOV2 indicate a universal dioxygenase mechanism utilized by CCOs.

5.1.5. The requirement of O2 for the isomerization reaction by RPE65

Unlike most family members with oxidative activity, the O2 requirement for

RPE65-mediated isomerization had not been validated experimentally.

Interestingly, both O2 dependent and independent isomerizing mechanisms have been proposed. By taking advantage of the improved methods used for ACO, we tested the involvement of O2 in RPE65-mediated isomerization. Our deoxygenation and activity studies of RPE65 in its native environment (RPE microsomes) showed that removing O2 failed to significantly inhibit subsequent reactions. Therefore, our data provided clear experimental evidence that the isomerase activity of RPE65 is O2-independent. Further, our results validated the role of a Lewis acid of the iron cofactor in this CCO rather than a more common redox chemical function for oxidative cleavage as in other family members.

Interestingly, NinaB integrates both oxidative cleavage and isomerizing activity in a single CCO. As the only CCO gene identified in insects so far, NinaB is responsible for generating the visual chromophore of vitamin A3 and is involved in its subsequent trans-to-cis isomerization. Consistent with isotope labeling results on ACO and NOV2, recent studies suggested a similar dioxygenase mechanism associated with NinaB [128]. It would be of interest to investigate how a single CCO could be enzymatically competent for such dual activity, substrate cleavage and isomerization, and what role the iron cofactor plays during catalysis.

186

5.2. Future directions

As shown in Chapter 2, our in situ analysis of an ACO-substrate reaction mixture by Raman spectroscopy showed great promise for other spectroscopic studies. As the 4-His+3-Glu iron center produces distinct spectroscopic signals, we believed that it is possible to use such signals to characterize iron centers as well as reaction mechanisms. For example, high resolution Raman spectroscopy

(e.g., resonance Raman spectroscopy) could be applied to provide insight into whether a dioxetane or epoxide intermediate is formed during catalysis.

Furthermore, together with other high resolution spectroscopic tools (e.g., x-ray absorption), future studies could probe the conformational changes in the iron center during catalysis, which would test the theory of the carbon cation reaction mechanism (i.e., stabilization of the carbon cationic intermediate through a nearby aromatic residue). Our studies already showed that ACO and NOV2 can be expressed and purified to yield high concentrations of protein (typically above 1 mM) without any detectable aggregation or significant loss in activity, which is extremely important and guarantee the success of above spectroscopic investigations. With progress in these areas, we would obtain a much clearer picture of CCO catalytic mechanisms.

Activity studies of ACO and NOV2 in the presence of an electron-enriched reagent, for example ascorbate or TCEP, failed to enhance ACO or NOV2 activities. This feature contrasts with many other non-heme, iron-dependent dioxygenases which typically need extra electron donors for catalysis. In addition, uncertainties regarding how the iron cofactor interacts with a dioxygen molecule

187

as well as the substrate remain unclear. Therefore, more studies could be done to address these important issues. For example, Electron Paramagnetic Resonance

(EPR) spectroscopy would be very useful to probe the interaction details between iron and substrate and/or O2. In fact, our initial EPR studies on ACO revealed that the presence of substrate in the non-heme iron center facilitates the binding of NO, which is frequently used as an O2 surrogate in the study of metal-oxygen interactions. The binding model of substrate and NO and the resulting E-S-NO complex may reveal reaction details during CCO catalysis. We could test the hypothesis that the presence of substrate in the active site triggers the O2 binding and initiates the subsequent cleaving reaction.

Finally, though challenging, obtaining the co-crystal structure of CCO with substrate or a substrate analog would be extremely useful, as it would clarify many of the uncertainties regarding the molecular details of substrate binding and greatly advance our understanding of the CCO catalytic mechanism. To accomplish this objective, multiple strategies can be applied to ACO, including 1) screen by enzymatic activity for an appropriate detergent/lipid milieu that will support a higher carotenoid solubility, 2) crystallize ACO in the presence of retinoid or inhibitors that were identified in our study (e.g., retinoic acid or retinyl acetate), 3) modify substrates to increase their water solubility, 4) screen for different crystallization conditions and 5) crystallize ACO in the presence of organic solvents.

After numerous attempts to crystallize an ACO complex, we conclude that the hydrophobicity of the carotenoids may be essentially insurmountable. The inclusion of such hydrophobic molecules in a crystallization cocktail may never

188

exceed the solubility limit to give a clear electron density signal in the substrate binding region. In addition, carotenoids also have a high propensity to self- aggregate. Therefore, they would precipitate from a crystallization solution. To circumvent this issue of substrate hydrophobicity, NOV2 could serve as a CCO for determining complex structure, since its stilbene substrates, including resveratrol and piceatannol, are much more soluble compared to the carotenoids. Further, our biochemical characterization of NOV2 demonstrated that the presence of detergent is not required for enzymatic activity. Therefore, detergent may not be a necessary component for protein crystallization. These encouraging features could eliminate issues associated with ligand occupancy. Of significance, previous work by Asami et al [179] identified a fluoro-derived resveratrol that is a potent inhibitor of NOV2. Our ongoing work already has confirmed the potency of this fluoro- derivative of resveratrol. Therefore, the fluoro-resveratrol could be used in co- crystallization or a soaking experiment to generate the complex structure.

Given the strong inhibition of NOV2 by fluoro-resveratrol, adding a fluorine group at the C-C cleaving site in a carotenoid could be a strategy to inhibit CCO enzymes. For example, introducing a fluoro group into β-carotene at the C15 (or

C15’) site could result in the potent inhibition of BCO1 in mammals, which would be a quite useful compound for BCO1 functional study. Similarly, because of the same iron reaction center, it would be interesting to determine whether fluoro- retinal might act as a strong inhibitor of RPE65, which in turn could be a drug candidate for the treatment of such visual dysfunctions such as age-related macular degeneration. Furthermore, success in establishing this method of CCO

189

inhibition could greatly advance in vivo functional studies of CCOs. Considering the importance of CCOs in human and in plant biology, this inhibition paradigm also could be valuable for diverse pharmaceutical and agricultural usages.

190

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