Substrate Specificity and Reaction Mechanism of Vertebrate Carotenoid Cleavage

Home , Neoxanthin, Phaseic acid, Retinol isomerase, Strigolactone, Zeaxanthin epoxidase

Substrate specificity and reaction mechanism of vertebrate carotenoid cleavage

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

Carlo C. dela Seña

The Ohio State Biochemistry Program

The Ohio State University

2014

Dissertation Committee:

Earl H. Harrison, Ph.D., Adviser

Robert W. Curley, Jr., Ph.D.

Ross E. Dalbey, Ph.D.

Steven J. Schwartz, Ph.D

Carlo C. dela Seña

2014

Abstract

Carotenoids are yellow, orange and red pigments found in fruits and vegetables.

Some carotenoids can act as dietary precursors of vitamin A. Humans and other animals generate retinal (vitamin A aldehyde) from provitamin A carotenoids by oxidative cleavage of the central 15-15′ double bond by the enzyme β-carotene 15-15′-oxygenase

(BCO1). Another carotenoid oxygenase, β-carotene 9′-10′-oxygenase (BCO2), catalyzes the oxidative cleavage of the 9′-10′ double bond of various carotenoids to yield apo-10′- carotenals and ionones.

In this dissertation, we elucidate the substrate specificity of these two enzymes.

Recombinant His-tagged human BCO1 was expressed in Escherichia coli strain BL21-

Gold (DE3) and purified by cobalt ion affinity chromatography. The enzyme was incubated with various dietary carotenoids and β-apocarotenals, and the reaction products were analyzed by reverse-phase high-performance liquid chromatography (HPLC). We found that BCO1 catalyzes the oxidative cleavage of only provitamin A dietary carotenoids and β-apocarotenals specifically at the 15-15′ double bond to yield retinal. A notable exception is lycopene, which is cleaved by BCO1 to yield two molecules of acycloretinal. Previous studies have found lycopene to be unreactive with BCO1. Our results warrant a fresh look at acycloretinal and its alcohol and acid forms as possible

ii metabolites of lycopene. We also found that BCO1 does not react with 9-cis-β-carotene.

It has been previously suggested the 9-cis-retinoic acid, a ligand of retinoid X receptors

(RXR’s), is generated by BCO1 cleavage of 9-cis-β-carotene to 9-cis-retinal and subsequently oxidized to the acid. However, our results strongly argue against this.

Similarly, the substrate specificity of purified recombinant chicken BCO2 was also tested by incubating purified enzyme with the test substrates and analysis of the products by HPLC. Unlike BCO1, BCO2 reacts with full length provitamin A carotenoids as well as non-provitamin A carotenoids that contain 3-hydroxy ionone rings.

However, it does not react with lycopene and β-apocarotenals.

Carotenoid cleavage oxygenases (CCO’s) oxidatively cleave carotenoids to yield aldehydes and/or ketones. Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging. BCO1 has been thought to be a monooxygenase based on a study that used conditions that favored oxygen exchange with water. We elucidated the reaction mechanism of BCO1 using the same principles of oxygen labeling experiments, but minimized the reaction and processing times to minimize oxygen exchange between retinal and water. We incubated purified

16 18 recombinant human BCO1 and β-carotene in an O2-H2 O medium for 15 minutes at

37°C, and the relative amounts of 18O-retinal and 16O-retinal were measured by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The 18O-retinal makes up only

3-10% of the total retinal. Incubation of 16O-retinal under the same conditions yields 5-

18 18 16 13% O-retinal. We also incubated BCO1 and β-carotene in an O2-H2 O medium.

Under these conditions, 18O-retinal makes up 79-85% of the total retinal product.

iii

Similarly, incubation of 91% 18O-retinal under the same conditions yields 67-84% 18O- retinal. Our results show that BCO1 incorporates only oxygen from O2 into retinal, and

BCO1 is therefore a dioxygenase.

Acknowledgments

I would like to express my deepest gratitude to my adviser, Dr. Earl H. Harrison.

Your guidance, support and encouragement made me grow as a scientist and as a person.

Thank you for believing in me and for always finding something good in my work, even during those times when I am disappointed in myself. I am eternally grateful for everything I learned from you.

Thanks to my committee members, Dr. Robert W. Curley Jr., Dr. Steven J.

Schwartz and Dr. Ross E. Dalbey, for their thoughtful comments that guided this work. I would also like to thank Dr. Curley and Dr. Schwartz for allowing me to work in their laboratories, and sharing their wisdom and resources. Thanks also to the members of the

Schwartz and Curley labs for attending to all my needs and inquiries.

I am grateful to Dr. Sureshbabu Narayanasamy for the excellent work on the synthesis of organic compounds for my projects, and to Dr. Kenneth M. Riedl for his outstanding expertise in mass spectrometry.

Thanks to the past and present members of the Harrison laboratory who worked with me- Dr. Matthew K. Fleshman, Dr. Abdulkerim Eroglu, Dr. Jian Sun, Vanessa

Reed, Shiva Raghuvanshi, Yan Yuan, and Sara Thomas. Thanks for helping me in the lab and for all the wonderful meals and conversations.

Special thanks to my first lab mates-Dr. Jason D. Fowler, Dr. Shanen Sherrer and

Dr. Cuiling Xu-for the training in the techniques that had been very useful to me in my research.

I am forever thankful to my friends in Columbus for being there for me through the best and worst of times. Thanks to my family for inspiring me to make the best of what I have.

Thanks to The Ohio State University and the United States of America for giving me this wonderful opportunity.

Vita

1999-2003 ...... B.S. Chemistry, Mindanao State University-Main Campus, Philippines 2003-2005 ...... Instructor, University of the Philippines at Los Baños, Philippines 2005-2006 ...... Chemist, Central Analytical Laboratory, San Miguel Corporation-Beer Division, Philippines 2006-2008 ...... M.S. Chemistry Program (completed 24 units), De La Salle University-Manila, Philippines Lecturer (part-time), De La Salle University-Manila, University of the Philippines-Manila, Manila, Philippines 2008-2009, 2010-2014 .. Graduate Research Associate, The Ohio State University 2009-2010 ...... Graduate Teaching Associate, The Ohio State University 2013-2014 ...... Food Innovation Center Doctoral Research Grant 2013 ...... Poster Award Winner, Carotenoids Research Interaction Group (CARIG)/Vitamin A Research Interaction Group (VARIG) Poster Competition

Publications

Eroglu, A., Hruszkewycz, D. P., dela Seña, C., Narayanasamy, S., Riedl, K. M., Kopec, R. E., Schwartz, S. J., Curley, R. W., Jr., and Harrison, E. H. (2012) Naturally-occurring eccentric cleavage products of provitamin A β-carotene function as antagonists of retinoic acid receptors. J. Biol. Chem. 287, 15886-15895

Harrison E.H., dela Seña, C., Eroglu A., Fleshman M.K. (2012) The formation, occurrence, and function of β-apocarotenoids: β-carotene metabolites that may modulate nuclear receptor signaling. Am. J. Clin. Nutr. 96:1189S-92S dela Seña, C., Narayanasamy, S., Riedl, K. M., Curley, R. W. Jr. Schwartz, S. J., and Harrison, E. H. (2013). Substrate specificity of purified recombinant human β-carotene 15,15'-oxygenase (BCO1). J. Biol. Chem. 288, 37094-37103

vii dela Seña, C. Riedl, K. M., Narayanasamy, S., Curley, R. W. Jr. Schwartz, S. J., and Harrison, E. H. (2013). The human enzyme that converts dietary provitamin A carotenoids to vitamin A is a dioxygenase. J. Biol. Chem. Published online March 25, 2014

Field of Study

Major Field: Biochemistry

viii

Table of Contents

Abstract ...... ii

Acknowledgments ...... v

Vita ...... vii

Table of Contents ...... ix

List of Abbreviations ...... xii

List of Tables ...... xiv

List of Figures...... xv

Chapter 1: Literature Review ...... 1

1.1 Introduction ...... 2

1.2 Strucure and metabolism of dietary carotenoids ...... 2

1.3 Carotenoid cleavage oxygenases...... 5

1.4 β-Carotene 15-15′-oxygenase ...... 10

1.5 β-Carotene 9′-10′-oxygenase ...... 13

1.6 Tables ...... 16

1.7 Figures ...... 19 ix

Chapter 2: Substrate specificity of purified recombinant human BCO1 ...... 26

2.1 Introduction ...... 28

2.2 Materials and Methods...... 30

2.3 Results ...... 36

2.4 Discussion ...... 39

2.5 Figures ...... 46

Chapter 3: Substrate specificity of purified recombinant chicken BCO2 ...... 54

3.1 Introduction ...... 56

3.2 Materials and Methods...... 57

3.3 Results ...... 61

3.4 Discussion ...... 63

3.5 Table ...... 68

3.6 Figures ...... 69

Chapter 4: The human enzyme that converts dietary provitamin A carotenoids to vitamin

A is a dioxygenase ...... 83

4.1 Introduction ...... 84

4.2 Materials and Methods...... 86

4.3 Results ...... 91

4.4 Discussion ...... 93

4.5 Table ...... 95

4.6 Figures ...... 96

Bibliography ...... 100

List of Abbreviations

ACO ...... apocarotenoid 15-15′ oxygenase

APcI...... atmospheric pressure chemical ionization

BCDO2 ...... β-carotene 9′-10′-dioxygenase

BCMO1 ...... β-carotene 15-15′-monooxygenase

BCO1 ...... β-carotene 15-15′-oxygenase

BCO2 ...... β-carotene 9′-10′-oxygenase

CCD ...... carotenoid cleavage dioxygenase

CCO ...... carotenoid cleavage oxygenase

CD36 ...... cluster determinant type 36

COBALT ...... Constraint-based Multiple Protein Alignment Tool cps...... counts per second

E.coli ...... Escherichia coli

EC ...... Enzyme Commission

HLADH ...... horse liver alcohol dehydrogenase

HPLC ...... high-performance liquid chromatography

IPTG ...... isopropyl β-D-1-thiogalactopyranoside kcat/KM ...... catalytic efficiency

xii

KM ...... Michaelis-Menten constant

LC-MS ...... liquid chromatography-mass spectrometry

LC-MS/MS ...... liquid chromatography-tandem mass spectrometry mAU ...... milli absorbance units

MRM ...... multiple reaction monitoring

MTBE ...... methyl t-butyl ether

NCED ...... 9-cis-epoxycarotenoid dioxygenase

NPCIL1 ...... Niemann-Pick C1 Like 1

Q-TOF ...... quadrupole time-of-flight

RPE ...... retinal pigment epithelium

RPE65 ...... retinal pigment epithelium protein of 65 kDa

RXR ...... retinoid X receptor

SD ...... standard deviation

SDS-PAGE ...... sodium dodecyl sulfate-polyacrylamide gel electrophoresis

SR-BI ...... scavenger receptor BI

UHPLC-MS/MS .... ultra-high-pressure liquid chromatography-tandem mass spectrometry

Vmax ...... maximum reaction velocity

VP14...... Viviparous 14

λmax ...... wavelength of maximum absorption

xiii

List of Tables

Table 1.1. Biochemical properties of recombinant BCO1 and BCO2 with β-carotene as

substrate...... 16

Table 1.2. Activity of recombinant BCO1 and BCO2 with substrates other than β-

carotene ...... 17

Table 1.3. Expression of carotenoid cleavage enzymes in different tissues in human and

mouse ...... 18

Table 3.1. Comparison of assay components used for BCO1 and BCO2 in vitro activity

assays ...... 68

Table 4.1. Comparison of retinal quantification by MS and MS/MS ...... 95

xiv

List of Figures

Figure 1.1. The carotenoid biosynthesis pathway in flowering plants ...... 19

Figure 1.2. The chemical structures of major dietary carotenoids ...... 20

Figure 1.3. Enzymatic oxidative cleavage of β-carotene ...... 21

Figure 1.4. Amino acid sequence similarity tree of CCO’s...... 22

Figure 1.5. Molecular phylogenetic tree of of the RPE65/BCO1/BCO2 family ...... 23

Figure 1.6. Homology comparison among BCO1, BCO2, RPE65 and other CCO’s...... 24

Figure 1.7. Schematic representation of the retinoid visual cycle ...... 25

Figure 2.1. Purification of recombinant human BCO1 using cobalt affinity column

chromatography...... 46

Figure 2.2. Kinetic data for purified recombinant BCO1 with β-carotene at 37°C ...... 47

Figure 2.3. Purified recombinant BCO1 cleaves β-carotene solely at the central 15-15′

bond ...... 48

Figure 2.4. Purified recombinant BCO1 cleaves lycopene to produce acycloretinal ...... 49

Figure 2.5. Substrate-velocity plots for other substrates that display Michaelis-Menten

behavior ...... 50

Figure 2.6. Substrate-velocity plots for β-apocarotenals ...... 51

Figure 2.7. Kinetic data for purified recombinant BCO1 and various carotenoids and

apocarotenals ...... 52

Figure 2.8. Purified recombinant human BCO1 does not cleave 9-cis-β-carotene ...... 53

Figure 3.1. Production of galloxanthin from zeaxanthin or lutein by BCO2 cleavage ..... 69

Figure 3.2. Sequence alignment of the isoforms of human BCO2 ...... 70

Figure 3.3. Sequence alignment of the isoforms of chicken BCO2 using COBALT ...... 71

Figure 3.4. Cleavage of β-carotene by purified recombinant chicken BCO2 ...... 72

Figure 3.5. Cleavage of α-carotene by purified recombinant chicken BCO2 ...... 74

Figure 3.6. Cleavage of β-cryptoxanthin by purified recombinant chicken BCO2 ...... 76

Figure 3.7. UV-Vis spectra of β-apo-8′-carotenal, β-apo-10′-carotenal, and their putative

3-hydroxylated counterparts produced from the reaction of chicken BCO2 with β-

cryptoxanthin ...... 78

Figure 3.8. Cleavage of lutein by purified recombinant chicken BCO2 ...... 79

Figure 3.9. Cleavage of zeaxanthin by purified recombinant chicken BCO2 ...... 81

Figure 3.10. Cleavage of carotenoids by murine BCO2 at the 9-10 and 9′-10′ bonds to

yield rosafluene dialdehyde ...... 82

Figure 4.1. Human BCO1 is a dioxygenase ...... 96

Figure 4.2. LC-MS chromatograms for the reaction mixture of BCO1 and β-carotene in

16 18 18 16 O2-H2 O and O2-H2 O ...... 98

Figure 4.3. MS/MS traces for the fragmentation of 16O-retinal and 18O-retinal obtained

18 16 from the reaction of BCO1 and β-carotene in O2-H2 O ...... 99

xvi

Chapter 1: Literature Review

1.1 Introduction

Vitamin A deficiency is a public health problem in poorer countries, especially in

Africa and Southeast Asia. According to the World Health Organization, an estimated

250,000 to 500,000 vitamin A deficient children become blind every year, half of them dying within 12 months of losing their sight (1). In chronically undernourished populations, maternal vitamin A deficiency also increases the risk of maternal morbidity and mortality (2). Humans and other animals cannot synthesize vitamin A and have to obtain it from dietary sources. Among the strategies being applied to address vitamin A deficiency is increasing consumption of carotenoid-rich foods (3).

Carotenoids are yellow, orange and red pigments synthesized by bacteria(4), microalgae (4), plants (5), and fungi (6). The carotenoid biosynthesis pathway in plants

(5) is shown in Figure 1.1. The characteristic yellow and orange colors of many fruits and vegetables are due to the presence of carotenoids. There are about 600 carotenoids in nature, and about 50 possess vitamin A activity, although far fewer are considered to have dietary importance (2). Various carotenoids, including those that do not have provitamin A activity, have also been shown to have antioxidant (7), anticancer (8) and photoprotective (9) activities, and their metabolites affect several cellular signaling pathways (8).

1.2 Strucure and metabolism of dietary carotenoids

The structures of major dietary carotenoids and the carbon numbering scheme are shown in Figure 1.2. Provitamin A carotenoids are those that yield retinal (vitamin A 2 aldehyde) by oxidative cleavage of the central bond (Figure 1.3). β-Carotene is the most abundant carotenoid in food and has the highest provitamin A activity (10). In vitro studies show that β-cryptoxanthin has 55% of the provitamin A activity of β-carotene, and α-carotene has 29% (11).

Most carotenoids found in raw fruits and vegetables are in the all-trans-form, but food processing increases the amount of cis isomers (10,12). For β-carotene, zeaxanthin and lutein, the main cis isomers found in processed food are the 9-cis and 13-cis forms

(10,13-14). In human plasma, a majority of these carotenoids are in the all-trans form

(14-16). It has also been suggested that dietary 9-cis-β-carotene isomerizes to the all- trans-form in the gastrointestinal lumen (17-18).

Lutein and zeaxanthin do not yield retinal by oxidative cleavage of the central bond and thus do not have provitamin A activity. Lutein and zeaxanthin are the only dietary carotenoids present in the macula of the human eye and their plasma levels are associated with a decreased risk of age-related macular degeneration (19).

Mesozeaxanthin, which differs from zeaxanthin only by the stereoisomeric configuration at the 3′ carbon, is thought to be generated in the retina from zeaxanthin; the mechanism has not been elucidated yet (20-21). Lutein, zeaxanthin and mesozeaxanthin are thought to protect the eye by absorbing actinic blue light and serving as antioxidants and free radical scavengers (22).

Lycopene is also a nonprovitamin A carotenoid that has garnered a great interest because it is associated with a decreased risk of prostate cancer (23). A significant proportion of lycopene (25-70%) in human body fluids and tissues are various cis

3 isomers (12). A human feeding study by Richelle et al. suggests that the 5-cis-isomer is absorbed better and/or does not isomerize significantly during uptake by the enterocytes, whereas about 40% of all-trans-lycopene is isomerized to various cis forms (24). The same study also suggests that the 9-cis and 13-cis isomers are isomerized to all-trans and

5-cis forms upon absorption. Three unidentified cis isomers of lycopene showed the highest antioxidant activity in vitro compared to all-trans-lycopene and all-trans-β- carotene (25).

Dietary carotenoids are incorporated into mixed micelles in the intestinal lumen, and absorbed by enterocytes. Early studies suggested that this absorption occurs by passive diffusion, but several proteins have been identified to facilitate the uptake of carotenoids (18). Scavenger receptor BI (SR-BI) (26-30), cluster determinant type 36

(CD36) (29-31) and Niemann-Pick C1 Like 1 (NPCIL1) (26,29,32) have been shown to be involved in the absorption of β-carotene, α-carotene and β-cryptoxanthin. SR-BI has also been shown to be involved in lutein and lycopene absorption (29,32). NPCIL1 is also involved in lutein and zeaxanthin absorption (26,32); its role in lycopene absorption is still debated (18).

In the enterocyte, provitamin A carotenoids are partially converted to retinal

(vitamin A aldehyde) by oxidative cleavage of the central 15-15′ double bond by the enzyme β-carotene 15-15′-oxygenase (BCO1) (Figure 1.3). Retinal can either be converted to retinol and subsequently to retinyl esters, or to retinoic acid (33). The retinyl esters and carotenoids are then incorporated into chylomicrons and secreted into the lymph (33).

In the general circulation, chylomicrons are converted to chylomicron remnants.

Most of the chylomicron remnants are cleared by the liver, which is a major storage site of retinyl esters and carotenoids (34-35). Further BCO1 cleavage of provitamin A carotenoids into retinal also occurs in the liver, especially in the hepatic stellate cells (34).

Extrahepatic uptake of carotenoids from chylomicron remnants may also be an important mode of delivery of carotenoids to other tissues (35). Tissues that tend to accumulate relatively high amounts of vitamin A (liver, adrenal glands, testis, adipose tissue) also accumulate carotenoids, and the opposite is true for tissues with low vitamin

A (kidney, lung) (36-37). In cases of hypercarotenemia, where carotenoids accumulate due to excessive dietary intake or a mutation in BCO1, a yellow-orange pigmentation is manifested in the skin, sweat and sebum (38-39).

1.3 Carotenoid cleavage oxygenases

The cleavage products of carotenoids (apocarotenoids) serve a variety of functions including pigments, aroma compounds and regulatory molecules (40).

Evidence of a carotenoid cleavage oxygenase (CCO) was first shown in 1965 by the formation of retinal from β-carotene using cell-free rat intestinal homogenate (41-42).

The first CCO to be cloned and characterized is maize Viviparous 14 (VP14), which cleaves 9-cis-epoxycarotenoids to yield the plant hormone abscisic acid (ABA in Figure

1.1) (43). This discovery ultimately led to the identification of more than 200 putative

CCO’s in sequence databases by sequence homology (44).

Most of the functionally characterized CCO’s are from plants. The comprehensive review by Walter et al. (40) identified five main clades of plant CCO’s: CCD1, CCD4,

CCD7, CCD8 and NCED (Figure 1.4), and a smaller group that cluster outside CCD4 that they named CCD4-related. Enzymes in the CCD4 and CCD7 clade and most of the functionally characterized CCD1 enzymes cleave a variety of carotenoids at the 9-10 and/or 9′-10′ bond (45-57). The CCD4-related enzymes and some of the CCD1 enzymes cleave at 5-6 and 5′-6′ bonds, and 7-8 and 7′-8′ bonds (58-60). The CCD8 enzymes cleave β-apo-10′-carotenoids at the 13-14 bond (50,61). The NCED enzymes cleave only

9-cis-carotenoid substrates (43,62). The range of cleavage positions also reflects the variety of functions of the cleavage products- aroma compounds (e.g. ionones from 9′-10′ cleavage), flavor compounds (e.g. safranal from 7′-8′ cleavage) and signaling molecules

(abscisic acid from 11-12 cleavage and strigolactones from 13-14 cleavage) (40).

CCO’s have been characterized in microorganisms. The CCO’s from

Synechocystis sp. PCC 6803 (63) and Nostoc sp. PCC7120 (64) both form retinal exclusively from β-apocarotenoids. A β-carotene 15-15′-oxygenase from a marine bacterium has also been characterized (65). A 13-14 apocarotenoid oxygenase has been identified in Novosphingobium aromaticivorans, producing β-apo-13-carotenone (66). A carotenoid oxygenase that can cleave at the 15-15′ and 13-14 bond has been identified in

Mycobacterium tuberculosis, the causative agent of tuberculosis (67), producing both retinal and β-apo-13-carotenone. Our laboratory has previously identified β-apo-13- carotenone as a potent antagonist of retinoid receptors (68-69), and the production of β-

6 apo-13-carotenone by a known pathogenic agent may be among its mechanisms for causing disease.

NinaB is the only CCO found in insects such as Drosophila melanogaster,

Anopheles gambiae, and Apis mellifera (70). Interestingly, purified recombinant Galleria mellonella NinaB shows both oxygenase and isomerase activities, catalyzing both the oxidation of the central 15-15′ bond of various all-trans-carotenoids to produce all-trans- and 11-cis-retinoids (70). In insects, three different 11-cis-retinoids have been identified to act as the visual chromophore depending on the species- retinal, (3R)-3-hydroxyretinal and (3S)-3-hydroxyretinal (71). Galleria mellonella NinaB can cleave different carotenoids that can yield all three of these retinoids but only zeaxanthin and lutein were found in the pupal heads (70), suggesting that the discrimination in visual chromophores stems from the discrimination in carotenoid absorption. In Drosophila, two members of the class B scavenger receptors, NinaD and Santa Maria, have been suggested to mediate carotenoid uptake in the gut and extra-retinal neural cells (72-73).

Similar to NinaB, RPE65 (retinal pigment epithelium protein of 65 kDa) is a member of the CCO family that possesses all-trans to 11-cis isomerase activity (74-78).

Unlike NinaB, RPE65 does not oxidatively cleave carotenoids (79) and thus, strictly speaking, cannot be called a CCO. Instead, RPE65 hydrolyzes all-trans-retinyl esters to produce 11-cis-retinol (74-78). RPE65 can also produce the 13-cis isomer, depending on the type of retinoid-binding proteins present (80-81). RPE65 is expressed in humans and other animals (Figs. 1.5 and 1.6). It is one of the three members of the CCO family encoded by the mammalian genome, along with BCO1 and β-carotene 9′-10′-oxygenase

(BCO2). Analogous to NinaB, RPE65 is part of the retinoid visual cycle in humans, forming the 11-cis retinoid that is ultimately photoisomerized to the all-trans-form (Fig

1.7). RPE65 has been identified as a membrane-binding protein associated with the smooth endoplasmic reticulum, although whether it is an integral or peripheral protein is still unclear (reviewed by Kiser and Palczewski (82)).

As of the time of writing, there are only three members of the CCO family with published crystal structures: the apocarotenoid 15-15′-oxygenase (ACO) from the cyanobacterium Synechocystis sp. PCC 6803 (83); bovine RPE65 (84); and maize VP14

(85). These three structures share the following features: a seven bladed β-propeller tertiary structure, a hydrophobic tunnel that leads to the active site and presumably interacts with the hydrophobic carotenoid, and an active site with four conserved histidine residues (86). The size of the hydrophobic tunnel in the apocarotenoid 15-15′- oxygenase from the cyanobacterium Synechocystis sp. PCC 6803 cannot accommodate a

β-ionone ring, which explains why it can only react with apocarotenoids but not β- carotene (83). Site-directed mutagenesis studies also show that specific aromatic and hydrophobic amino acid residues in the hydrophobic tunnel, rather than the active site, influence the ratio of 11-cis-to 13-cis-retinol isomer produced by RPE65 (81,87-88).

Removal of the methyl group at the C13 and/or C13′ of β-carotene significantly reduces its activity with chicken BCO1, suggesting important interactions with hydrophobic groups in the substrate tunnel (89). Additional hydrophobic tunnels connected to the substrate tunnel at the catalytic center (three in Synechocystis ACO and two in maize

VP14) could also potentially serve as exit tunnels for the cleavage products (90).

The four conserved histidine residues bind ferrous ion. The iron-binding residues have been also verified by site-directed mutagenesis (76,91-92). Iron-chelating agents have also been shown to inhibit various CCO’s and RPE65 (43,84,93-96). The iron in

CCO’s is thought to activate molecular oxygen for cleavage of carbon-carbon double bonds to aldehydes, similar to other alkene cleavage oxygenases (83,97-98). For RPE65, the role of iron is not clear. In their review of RPE65, Kiser and Palczewski speculated that the iron in RPE65 interacts directly with carbonyl oxygen of the all-trans-retinyl ester to form a carbocation intermediate, which is then stabilized by the aromatic residues lining the tunnel leading to the iron center (82). The same group later crystallized RPE65 from an enzymatically active retinal pigment epithelium (RPE) microsomal extract, and found that the iron is coordinated to the carboxylate group of a fatty acid, supporting their previous hypothesis (99).

There is limited information on the specificity of CCO’s for Fe2+. Other metals such as Ni2+ and Zn2+ have been shown to be able to replace Fe2+ in other non-heme iron oxygenases (100-101). After treatment with o-phenanthroline (an iron-chelating agent), the β-carotene 15-15′-oxygenase from a marine bacterium showed significant activity rescue with Fe3+, and Co2+, and very little to no activity rescue with Mg2+, Mn2+, Ba2+,

Ca2+and Cu2+ (65). For RPE65, the inhibition of activity by iron chelators can only be restored by addition of FeSO4, but not by CuSO4, ZnCl2, MgCl2, Fe (III) citrate or FeCl3, indicating that Fe2+ is the metal ion essential for the isomerohydrolase activity (96). The cleavage of β-carotene to crocetindial and β-cyclocitral by Microcystis PCC 7806 cells is stimulated by Fe2+, Fe3+, and Ca2+ (102).

1.4 β-Carotene 15-15′-oxygenase

(This section was adapted from a book chapter currently in press submitted by the author and Earl H. Harrison (86))

Early studies on carotenoid metabolism in animals are focused on provitamin A carotenoids and their enzymatic conversion to vitamin A. The first in vitro studies published in 1965 used extracts from rat liver and intestinal mucosa to demonstrate enzymatic oxidative cleavage of β-carotene at the central double bond to yield retinal

(41-42). In 2000, the enzyme responsible for the conversion of β-carotene to retinal, β- carotene-15-15′-oxygenase (BCO1, also BCMO1), was identified in Drosophila (79) and chicken (103). BCO1 has also been identified in humans (93,104-105), mice (94,106), rat

(107), cows (108), zebrafish (109) and Caenorhabditis elegans (110) .

The study of carotenoid oxygenase enzymes is complicated by the fact that carotenoids are also prone to nonenzymatic oxidation. Oxidation products may be produced nonenzymatically during incubation, extraction and processing. Maret and

Hansen incubated β-carotene under conditions similar to the early in vitro studies of

Goodman and Olson in the absence of enzyme and detected eccentric cleavage products or β-apocarotenoids (111). Tang et al. and Wang et al. reported the production of β- apocarotenoids and retinal from the incubation of β-carotene with intestine homogenates

(112-113). However, the same group later reported that retinal is the sole oxidative cleavage product if the in vitro incubations are done with α-tocopherol (114). The elimination of eccentric cleavage products by α-tocopherol, an antioxidant, suggests that they were formed from chemical oxidation.

Intestinal homogenates were used in most of the early in vitro studies as a source of BCO1. However, the intestine also contains peroxidases that can potentially oxidize carotenoids (111). Thus, a no-enzyme control or a heat-denatured enzyme control will not be able to differentiate between enzymatic oxidation by BCO1 and other enzymes present in intestine homogenates. To clearly elucidate the properties of BCO1 and BCO2, a purified enzyme preparation is imperative. The identification of the genes for BCO1 and BCO2 paved the way for the production of purified recombinant enzyme. In the following discussion, the in vitro studies cited are mostly limited to purified and/or recombinant enzyme.

The biochemical properties of recombinant BCO1 are summarized in Table 1.1.

Through site-directed mutagenesis experiments, it has been shown that the ferrous ion in

BCO1 is bound by four conserved histidine residues and one glutamate residue (91)

Consequently, enzyme activity can be inhibited by ferrous ion chelators such as α,α′- dipyridyl and o-phenanthroline (93-95). BCO1 is also inhibited by sulfhydryl alkylating agents (e.g. N-ethylmaleimide and p-chloromercuribenzoate) and stimulated by reducing agents (e.g. reduced glutathione and dithiothreitol) (93). This suggests the importance of reduced cysteines. There are six conserved cysteines in human, mouse, rat and chicken

BCO1 isozymes (93), but their roles have not yet been elucidated.

A monooxygenase mechanism has been proposed for BCO1 (115). In this study,

α-carotene, purified chicken BCO1 and horse liver aldehyde dehydrogenase were

17 18 incubated in an 85% O2-95% H2 O environment. The resulting products (retinol and α- retinol) were purified by HPLC and silylated. Using gas chromatography-mass

11 spectrometry (GC-MS), they found virtually equal enrichment of 17O and 18O in both silylated retinols, suggesting a monooxygenase mechanism for BCO1. However, it is possible that the long reaction time (7.5 hours) and extensive processing (reduction of the aldehydes, purification, silylation) favored oxygen exchange between the initial aldehyde products and the medium. A computational study of the reaction mechanism of ACO predicted the dioxygenase mechanism (98). Despite this controversy, animal orthologs of

BCO1 have been called β-carotene 15-15′-monooxygenase, and the corresponding gene has been named BCMO1 (116).

β-Carotene has been shown to be the best substrate for BCO1 (93,117). BCO1 only reacts with carotenoids with at least one unmodified ionone ring and at least 30 carbons (Table 1.2). BCO1 activity with lycopene has been reported for the mouse homolog (106), but the human and chicken homologs have been reported to be unreactive

(93,117).

BCO1 is a soluble (cytosolic) enzyme (93) and exists as a monomer (39,118). It is expressed in a variety of tissues (Table 1.3). Early experiments have shown high BCO1 activity in tissue homogenates from intestines (41-42,119) and it is no surprise that BCO1 expression was detected in intestine tissues using immunostaining and RNA blot. The wide variety of tissues where BCO1 is expressed suggests a greater role for BCO1 than just breaking down carotenoids after a meal.

The most obvious function of BCO1 is to generate retinal (vitamin A aldehyde) from dietary carotenoids. Indeed, BCO1 KO mice tend to have high levels of stored carotenoids and low levels of retinol and retinyl esters (120). A loss-of-function mutation

12 in BCO1 has been identified in a patient with hypercarotenimia and hypovitaminosis A

(39). However, BCO1 KO mice have been shown to develop liver steatosis, elevated free fatty acids and obesity even on a vitamin A sufficient diet (120-121). This suggests a greater role for BCO1 than merely generating retinal.

A significant portion of carotenoids in the diet are not provitamin A, such as zeaxanthin, lutein and lycopene (122). Zeaxanthin and lutein have very similar structures to β-carotene and can possibly compete for binding with BCO1. Zeaxanthin and canthaxanthin have been shown to inhibit β-carotene cleavage both in vitro and when fed to rats (123). Other dietary molecules that might have enough similarity in shape to carotenoids also inhibit BCO1 activity, such as flavonoids. The structure-activity relationships observed in inhibition of BCO1 activity by flavonoids indicate that the catechol structure of ring B in flavonol is essential for inhibition (124).

1.5 β-Carotene 9′-10′-oxygenase

(This section is adapted from a book chapter currently in press submitted by the author and Earl H. Harrison (86))

In 2001, another carotenoid cleavage enzyme that catalyzes excentric cleavage, β- carotene-9′, 10′-oxygenase (BCO2), was identified in human, mouse and zebrafish (125).

It is interesting to note that although there has been no oxygen labeling experiments done with BCO2, the animal orthologs of the enzyme have been called either a dioxygenase

(126) or monooxygenase (127) in literature, but the corresponding gene is still called

BCO2 (128). This is in contrast to BCO1, as discussed in the previous section. The 13 biochemical properties of recombinant BCO2 are summarized in Table 1. At this point, we cannot compare the catalytic efficiencies of BCO1 and BCO2 against β-carotene because of the different assay systems used by the studies cited above. For example, the human BCO1 study by Lindqvist and Andersson (93) used 1-S-octyl β-D- thioglucopyranoside micelles to deliver the carotenoid substrates, while the human BCO2 study by Kim et al. (129) used Tween-40.

Unlike BCO1, BCO2 is able to catalyze the oxidative cleavage of xanthophylls such as zeaxanthin and lutein (126,130). The kinetic parameters obtained by Mein et al. also suggest that zeaxanthin and lutein are better substrates than β-cryptoxanthin. Ferret

BCO2 is able to cleave 5-cis- and 13-cis-lycopene, but not the all-trans-form (127).

Kinetic data for different carotenoids with BCO2 are lacking, but the limited information available suggests that BCO2 has broader substrate specificity with respect to substrate molecule shape.

In contrast to BCO1, BCO2 is a mitochondrial enzyme, and has been suggested to function primarily in preventing oxidative stress due to carotenoid accumulation by breaking down excess carotenoids (126). The cleavage products of β-carotene with

BCO2, β-ionone and β-apo-10′-carotenal, have not been identified to have any activity.

These cleavage products have been reported to be inactive with respect to retinoic acid response element (RARE) transactivation assays (68).

BCO2 and BCO1 are expressed in a variety of tissues (Table 1.3). BCO1 but not

BCO2 is expressed in the colon, ovary, skin epidermis and brain. Also, the immunostaining for BCO1 is much stronger compared to BCO2 in the stomach and the

14 epithelium of the uterine glands of the endometrium (131). Conversely, BCO2 but not

BCO1 is expressed in pancreas islet cells, stroma of the prostate, stroma of the endometrium and cardiac tissue. The immunostaining for BCO2 is much stronger compared to BCO1 in skeletal muscle (131). These differences in expression suggest that

BCO2 may play roles that are distinct from those of BCO1.

BCO1 knockout (KO) mice have elevated expression of BCO2, and vice versa

(132-133). Upon β-carotene supplementation, BCO1 KO mice accumulate β-apo-10′- carotenol, the alcohol form of the β-carotene-BCO2 cleavage product (121). BCO2 KO mice accumulate 3,3′-didehydrozeaxanthin and 3-dehydrolutein upon supplementation with zeaxanthin and lutein, respectively (126). This is consistent with the findings of other groups that these xanthophylls are not substrates for BCO1. After a tomato- supplemented diet, lycopene accumulated in BCO2 KO mice compared to wild-type

(WT) (134). This suggests that lycopene is a substrate for BCO2. However, it has not been clearly established whether BCO2 catalyzes the cleavage of all-trans-lycopene or cis isomers.

1.6 Tables

BCO1 BCO2 Human Mouse Chicken (117) Human Mouse Ferret Molecular 63 (93,135), 64 (94), 61 63 (131), 60(125) 61 (127) mass, kDa 62 (136) 65 (106) 57 (129) pH optimum 7.5-8 (93) 8.0 8.0 (129) 8.0-8.5 (127) KM, μM 7 (93), 31 6 (106) 26 67 (129) 3.5 (127) (136), 18 (135) Vmax, nmol 600 (93), 2 (106) 32.2 cleavage 66,000 (127) product/mg/h (136), 138 (retinal for (135) BCO1, apo- 10′-carotenal for BCO2)

Table 1.1. Biochemical properties of recombinant BCO1 and BCO2 with β-carotene as substrate. Numbers in bold are the values of the parameters; numbers in parentheses and regular text are bibliographical reference numbers.

BCO1 BCO2 Human Mouse Chicken(117) Mouse Ferret 9-cis-β-carotene  (132)  (132) α-carotene  γ-carotene  ε-carotene  (106) All-trans-lycopene X (93)  (106) X X (127) 5-cis- and 13-cis-lycopene  (127) β-cryptoxanthin  (93)   (126)  (130) Zeaxanthin X (93) X  (126)  (130) Lutein X  (126)  (130) Canthaxanthin  (137) β-apo-4′-carotenal  β-apo-8′-carotenal  β-apo-12′-carotenal X -The substrate is oxidatively cleaved by the enzyme X-no reaction

Table 1.2. Activity of recombinant BCO1 and BCO2 with substrates other than β- carotene.

BCO1 BCO2 Human Mouse Human Mouse (104-105) (94,106,125) (131) (125) Stomach  X  X Small intestine     Colon  X X X Liver     Pancreas Acinar cells   Islet cells X  Kidney     Testis     Ovary  X Adrenal gland   Prostate Epithelium of glands   Stroma X  Endometrium   Epithelium of uterine glands   Stroma X  Skeletal muscle  X  Skin epidermis   X Retinal pigment epithelium   Brain  X X  Retina X Heart X X   Placenta X X Lung X X X  Spleen X X X  Peripheral blood leukocytes X X

-Expression is detected by either immunostaining or RNA blotting X-No expression detected

Table 1.3. Expression of carotenoid cleavage enzymes in different tissues in human and mouse.

1.7 Figures

Figure 1.1. The carotenoid biosynthesis pathway in flowering plants. Enzymes are named according to the designation of their genes. The pathway in the box takes place in chromoplasts of pepper fruit. A, 4-diphosphocytidyl-2C-methyl-D-erythritol; B, 4- diphospho-cytidyl-2C-methyl-D-erythritol 2-phosphate; C, 2C-methyl-D-erythritol 2,4- cyclodiphosphate; ABA, abscisic acid; AO, adldehyde oxidase; Ccs, capsanthin- capsorubin synthase; CrtR-b, β-ring hydroxylase; CrtR-e, ε-ring hydroxylase; Cyc-b, chromoplast-specific lycopene cyclase; DMADP, dimethylallyl diphosphate; DOXP, 1- deoxy-D-xylulose 5-phosphate; DPME, 4-diphosphocytidyl-2C-methyl-D-erythritol; DXR, DOXP reductoisomerase; DXS, DOXP synthase; GA3P, glyceraldehyde-3- phosphate; GGPP, geranylgeranyl diphosphate; Ggps, GGPP synthase; Ipi, IPP isomerase gene; IPP, isopentenyl diphosphate; ispD, DPME synthase; ispE, DPME kinase; ispF, 2C-methyl-D-erythritol 2,4-cyclodiphosphate; Lcy-b/Crtl-b, lycopene β-cyclase; Lcy- e/Crtl-e, lycopene ε-cyclase; MEP, 2-C-methyl-D-erythritol 4-phosphate; Nxs, neoxanthin synthase gene; Pds, phytoene desaturase; Psy, phytoene synthase; Vde1, violaxanthin deepoxidase 1; VNCED (VP14), 9-cis-epoxycarotenoid dioxygenase; Zds, ζ-carotene desaturase; Zep1, zeaxanthin epoxidase?1. From Hirschberg (5). 19

β-carotene

α-carotene

β-cryptoxanthin

Zeaxanthin

Lutein

Lycopene

Figure 1.2. The chemical structures of major dietary carotenoids. The carbon numbering scheme is shown for β-carotene.

Figure 1.3. Enzymatic oxidative cleavage of β-carotene. Two enzymes have been identified to cleave β-carotene at specific positions. BCO1 cleaves β-carotene at the central double bond to yield two molecules of retinal, while BCO2 cleaves at the 9′-10′ bond.

Figure 1.4. Amino acid sequence similarity tree of CCO’s. The five clades of plant CCO’s are highlighted in color. The CCO’s from animals (human, mouse and Drosophila) and cyanobacteria (Nostoc and Synechocystis) are also shown. The tree has been generated from ClustalW alignments using the programs Distances and Splits of the Heidelberg Unix Sequence Analysis Resources (HUSAR) software package. Adapted from Walter et al. (40).

Figure 1.5. Molecular phylogenetic tree of the RPE65/BCO1/BCO2 family. The phylogenetic tree shows the similarities of different CCO’s from several vertebrates. Phylogenetic trees were inferred from amino acid sequences using the neighbor-joining method using ClustalX. The numbers at the nodes are bootstrap values based on 1000 replicates. Scale bar, 0.1 amino acid replacements per site. Adapted from Kusakabe et al. (138).

BCO1 Human Ferret Mouse Rat Bovine Sheep Chicken Zebrafish Ferret BCO1 85 Mouse BCO1 85 85 Rat BCO1 84 85 93 Bovine BCO1 81 83 77 77 Sheep BCO1 79 80 76 76 90 Chicken BCO1 69 68 71 71 66 65 Zebrafish BCO1 59 56 59 60 56 55 59 Human BCO2 38 38 37 37 36 34 43 37 Ferret BCO2 40 39 40 39 39 37 42 40 Mouse BCO2 40 40 42 40 41 39 42 38 Rat BCO2 39 40 40 40 40 39 42 38 Bovine BCO2 38 38 37 37 36 35 41 37 Sheep BCO2 36 38 38 36 35 34 39 37 Chicken BCO2 39 40 39 39 37 36 42 38 Zebrafish BCO2 38 39 40 39 38 37 39 39 Human RPE65 38 40 39 40 38 36 40 36 Bovine RPE65 38 39 39 39 38 36 40 36 Rat RPE65 38 40 40 40 39 36 40 35 Mouse RPE65 38 40 39 40 38 36 40 36 Chicken RPE65 38 39 39 39 38 35 39 36 Zebrafish RPE65 40 41 40 41 38 36 39 36 Drosophila NinaB 30 31 30 30 28 27 35 32 Arabidopsis CCD1 16 16 17 15 15 14 17 18 Maize VP14 15 15 15 13 13 12 17 15

BCO2 Human Ferret Mouse Rat Sheep Bovine Chicken Zebrafish Ferret BCO2 84 Mouse BCO2 81 80 Rat BCO2 80 80 92 Bovine BCO2 80 85 82 82 Sheep BCO2 78 81 75 75 91 Chicken BCO2 62 67 66 64 64 63 Zebrafish BCO2 55 58 58 57 56 54 59 Human RPE65 42 41 41 42 41 39 41 41 Bovine RPE65 41 41 41 42 41 38 41 41 Rat RPE65 42 42 41 43 42 39 41 42 Mouse RPE65 42 42 42 43 42 39 41 42 Chicken RPE65 42 42 42 43 42 39 42 42 Zebrafish RPE65 39 38 40 40 39 36 38 38 Drosophila NinaB 29 31 33 34 29 30 30 32 Arabidopsis CCD1 18 18 17 18 17 13 17 15 Maize VP14 17 17 18 18 17 15 17 15

RPE65 Drosophila Arabidopsis Human Bovine Rat Mouse Chicken Zebrafish NinaB CCD1 Bovine RPE65 98 Rat RPE65 94 93 Mouse RPE65 93 93 97 Chicken RPE65 90 89 87 88 Zebrafish RPE65 74 74 72 73 73 Drosophila NinaB 33 32 33 33 32 32 Arabidopsis CCD1 12 12 14 11 15 13 13 Maize VP14 15 15 15 15 14 15 9 35

Sequence homology key ≥90% 70-89% 40-69% <39%

Figure 1.6. Homology comparison among BCO1, BCO2, RPE65 and other CCO’s. Sequence homology generated by ClastalW2. Adapted from Lietz et al. (139)

Figure 1.7 Schematic representation of the retinoid visual cycle–Vision begins when light (hν) causes photoisomerization of the 11-cis-retinylidene chromophore of ground- state rhodopsin (Rho). Subsequently, the Schiff base linkage loses a proton enabling rhodopsin to activate G proteins (i). After remaining active for a short period of time, the isomerized chromophore is released via hydrolysis, generating free all-trans-retinal and opsin (ii). The all-trans-retinal is enzymatically reduced by a retinol dehydrogenase (RDH) (iii) and the resultant all-trans-retinol is exported from the rod outer segment to the RPE. Here all-trans-retinol is metabolized by lecithin:retinol acyltransferase (LRAT) to produce all-trans-retinyl esters (iv), which can be either stored in retinosomes or further processed. RPE65 is the key enzyme that catalyzes the conversion of all-trans- retinyl esters to 11-cis-retinol (v). 11-cis-Retinol is enzymatically reduced to 11-cis- retinal (vi), which is then transported back to the photoreceptor outer segment where it recombines with opsin to form ground-state rhodopsin (vii). Continuous operation of this cycle is what sustains vision under conditions where rods are primarily active. From Kiser and Palczewski (82).

Chapter 2: Substrate specificity of purified recombinant human BCO1*

*dela Sena, C., Narayanasamy, S., Riedl, K. M., Curley, R. W., Jr., Schwartz, S. J., and Harrison, E. H. (2013) Substrate specificity of purified recombinant human β-carotene 15,15'-oxygenase (BCO1). J. Biol. Chem. 288, 37094-37103

ABSTRACT

Humans cannot synthesize vitamin A and thus have to obtain it from their diet. β-

Carotene 15-15′-oxygenase (BCO1) catalyzes the oxidative cleavage of provitamin A carotenoids at the central 15-15′ double bond to yield retinal (vitamin A aldehyde). In this work, we quantitatively describe the substrate specificity of purified recombinant human

BCO1 in terms of catalytic efficiency values (kcat/KM). The full length open reading frame of human BCO1 was cloned into the pET28b expression vector with a C-terminal polyhistidine tag, and the protein was expressed in the Escherichia coli strain BL21-Gold

(DE3). The enzyme was purified using cobalt ion affinity chromatography. The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a Vmax = 197.2

-1 nmol retinal/mg BCO1 x hr, KM = 17.2 μM and catalytic efficiency kcat/KM = 6098 M min-1. The enzyme also catalyzed the oxidative cleavage of α-carotene, β-cryptoxanthin and β-apo-8′-carotenal to yield retinal. The catalytic efficiency values of these substrates are lower than that of β-carotene. Surprisingly, BCO1 catalyzed the oxidative cleavage of lycopene to yield acycloretinal with a catalytic efficiency similar to that of β-carotene.

The shorter β-apocarotenals (β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′- carotenal) do not show Michaelis-Menten behavior under the conditions tested. We did not detect any activity with lutein, zeaxanthin, and 9-cis-β-carotene. Our results show that

BCO1 favors full length provitamin A carotenoids as substrates, with the notable exception of lycopene. Lycopene has previously been reported to be unreactive with

BCO1, and our findings warrant a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.

2.1 Introduction

Carotenoids are yellow and orange pigments found in fruits and vegetables. They are a major dietary source of vitamin A. In humans and other animals, provitamin A carotenoids are converted to retinal (vitamin A aldehyde) via the oxidative cleavage of the central double bond by the enzyme β,β-carotene 15-15′-oxygenase (BCO1) (93-

94,103,106,109,117,136). Although it is clear that BCO1 cleaves primarily at the central double bond, the possibility that it can also perform eccentric cleavage has not been well studied.

BCO1 is expressed in several tissues, and high enzymatic activity has been shown for homogenates of liver and intestine (41-42,113,140-142). Early studies of substrate specificity of BCO1 used liver and intestine homogenates to show the enzymatic production of retinal from provitamin A carotenoids (β-carotene, α-carotene, β- cryptoxanthin) and β-apocarotenals (β-apo-8′-, β-apo-10′- and β-apo-12′-carotenal)

(89,119,123,143). These studies also show that xanthophylls (lutein, zeaxanthin and astaxanthin) are not cleaved by BCO1.

In raw fruits and vegetables, β-carotene exists predominantly in the all-trans- form, but processing leads to an increase of cis isomers, particularly 9-cis-and 13-cis- forms (10). These isomers have also been detected in human plasma (14,144). Cleavage activity with these β-carotene isomers have been shown by incubation with intestine and liver homogenates (123,145-146).

However, the use of cell homogenates to study enzymatic oxidation of carotenoids in vitro is problematic. Carotenoids are relatively easy to oxidize, and even

28 no-enzyme incubations carried out for relatively long periods (1 hour) with β-carotene produces random cleavage products (111). The intestine contains other enzymes that could potentially oxidize carotenoids (reviewed by Maret and Hansen) (111). Indeed, early studies with intestine homogenates have detected random cleavage products in addition to retinal (112-113). Although the use of an anti-oxidant such as α-tocopherol reduces the amount of random cleavage products (114), it is difficult to determine whether the enzymatic activity is modulated by the other components of the crude enzyme preparation.

The identification and cloning of BCO1 (79,103) paved the way for the production and purification of recombinant BCO1. Two studies have used purified recombinant BCO1 preparations to study substrate specificity, and for most part, agree with the suggestion of previous studies that BCO1 only cleaves carotenoids with at least one unsubstituted β-ionone ring (93,117). Specifically, both studies showed that purified recombinant BCO1 does not cleave lycopene. Yan et al., using extracts from Sf9 cells expressing recombinant human BCO1, also report that lycopene is not a substrate (105).

These studies are in contrast with the study of Redmond et al., who observed that expression of murine BCO1 in lycopene-accumulating E.coli results in bleaching of the red lycopene pigment, indicating cleavage of lycopene (106). Kim and Oh also showed that purified recombinant chicken BCO1 cleaves β-apo-8′-carotenal but not β-apo-12′- carotenal, suggesting that BCO1 will not cleave the shorter β-apo-14′-carotenal (117).

In this work, we investigated the activity of purified recombinant human BCO1 with major dietary carotenoids and β-apocarotenals. We show that purified recombinant

29 human BCO1 catalyzes the cleavage of lycopene to produce acycloretinal (apo-15- lycopenal), and β-apocarotenals as short as β-apo-14′-carotenal to produce retinal. We also find that it does not catalyze the cleavage of 9-cis-β-carotene.

2.2 Materials and Methods

Carotenoids and retinoids- β-Carotene (≥97%), all-trans-retinal (≥98%), 9-cis-retinal

(≥95%), 13-cis-retinal (≥85%), α-carotene (≥95%), β-cryptoxanthin (≥97%), zeaxanthin

(≥95%), lutein (≥90%) and β-apo-8′-carotenal (≥96%) were purchased from Sigma-

Aldrich. The lycopene standard (92% all-trans, 6% 5-cis, 2% other isomers) was a gift from Dr. Steven J. Schwartz of The Ohio State University. β-Apo-10′-carotenal, β-apo-

12′-carotenal and β-apo-14′-carotenal were synthesized according to published methods

(68). The synthesis of acycloretinal (apo-15-lycopenal) used similar methods and will be reported in a future publication. The structures of synthesized substrates and standards were confirmed by mass spectrometry, ultraviolet-visible spectrophotometry and nuclear magnetic resonance spectroscopy, and purity (≥95%) was further assessed by HPLC analysis. All experiments involving carotenoids and retinoids were done under amber lights.

Isolation of 9-cis-β-carotene from Betatene®- Betatene® is a supplement of carotenoids extracted from the alga Dunaliella salina that is known to have relatively high amounts of 9-cis-β-carotene (15). Five capsules of Betatene® (Swanson Vitamins) were sliced open under methanol, and the resulting slurry of carotenoids was transferred to a conical 30 glass centrifuge tube. The mixture was centrifuged and the residue was washed three times with 5 mL of ethanol per wash. The residue was transferred to a separatory funnel with 1:1 (v/v) hexanes-methanol. The volume of the mixture was then brought up to about 100 mL of 1:1 (v/v) hexanes-methanol, and 5 mL of water was added, producing two phases. The hexane layer was evaporated under argon, and the residue was dissolved in 75:25 (v/v) methanol-methyl t-butyl ether (MTBE). The sample was separated using

HPLC Method C described below. The 9-cis-β-carotene fraction was collected, evaporated under argon, and stored at -80°C until used. The product is about 95% pure by

HPLC.

Expression and purification of recombinant human BCO1- A pET-28b plasmid vector containing the cDNA of human BCO1 with a C-terminal hexahistidine tag was a gift from Dr. William Blaner of Columbia University. The plasmid was transformed into

E.coli BL21-Gold (DE3) (Stratagene) according to the manufacturer’s instructions. The transformed bacterial cells were grown in LB broth (Sigma-Aldrich) to an OD600 of 0.5-

0.7 at 30°C and expression of the recombinant protein was induced by adding isopropyl

β-D-1-thiogalactopyranoside (IPTG, Gold Biotechnology) to a final concentration of 0.1 mM. Five liters of culture were grown for 24 hours and the cells were harvested by centrifugation. The cell pastes were frozen at -80°C and thawed prior to lysis. The cells were lysed by gentle stirring with lysis buffer [50 mM phosphate, 50 mM NaCl, 10%

(v/v) glycerol, 1% (v/v) Triton X-100, 1 mM β-mercaptoethanol, 2.5 mM imidazole, 10 mg/mL chicken egg white lysozyme (Sigma), 0.1 μL/mL Benzonase Nuclease HC

(Novagen), 1 mM phenylmethanesulfonylfluoride and 1 tablet EDTA-free protease inhibitor cocktail (Roche)/50 mL], using 5 mL of lysis buffer per gram of wet cell paste.

The lysis was conducted in ice for 1 hour. The lysate was centrifuged at 15,600 x g for 25 minutes and the supernatant was filtered through a 0.22 μm syringe-driven filter. The

NaCl concentration of the filtered supernatant was brought up to 300 mM using salt adjustment buffer (50 mM phosphate, 2,500 mM NaCl, 10% (v/v) glycerol, 1% (v/v)

Triton X-100, 1 mM β-mercaptoethanol, 2.5 mM imidazole) and stirred with 0.5 mL of

HisPur resin (Pierce) that has been previously washed with equilibration buffer (50 mM phosphate, 300 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mM β- mercaptoethanol, 2.5 mM imidazole) for 1 hr in an ice bath. The resin was harvested by centrifugation at 700 x g for 2 minutes, and transferred to a gravity column. The resin was then eluted with the following solutions, which were prepared by combining the calculated amounts of equilibration buffer (2.5 mM imidazole) and elution buffer (same composition as equilibration buffer except 150 mM imidazole): 4 mL of 2.5 mM imidazole (equilibration buffer), 1.5 mL of 10 mM imidazole, 1 mL each of 30, 60, 90,

120 and 150 mM imidazole (elution buffer). The fractions with pure protein were transferred into the final storage buffer (50 mM phosphate, 50 mM NaCl, 10% (v/v) glycerol, 1% (v/v) Triton X-100, 1 mM β-mercaptoethanol) using a PD-10 desalting column (GE Healthcare). SDS-PAGE was used to assess the purity of the protein throughout the purification process. The concentration of the protein solution was measured using the reducing agent compatible-BCA Protein Assay Kit (Pierce). The protein was stored at -80°C until used.

In vitro BCO1 activity assay- The in vitro BCO1 enzyme assay was based on the method of During et al. (142), with the following exceptions. First, 500 μL of 1:1 (v/v) acetonitrile-isopropyl alcohol (instead of acetonitrile alone) was added after quenching the reaction with formaldehyde. Second, the resulting mixture was filtered through a 0.22

μM syringe filter, instead of centrifugation to remove the insoluble particles. Third, only

100 μL of the sample, instead of 200 μL as in the original method, was injected into the

HPLC. Five hundred ng of purified recombinant BCO1 are used per reaction, and the reaction time is 15 minutes, unless otherwise indicated. Standard curves were generated by dissolving various amounts of standard compounds (retinal or apo-15-lycopenal) in a solution of 200:0.3:50:250:250 (v/v/v/v) water-Tween-40-37% formaldehyde- acetonitrile-isopropyl alcohol, which has the same ratio of solvents as the samples obtained from the reaction mixtures.

The assay is sensitive to Triton X-100 concentration. We have found the optimum enzyme activity at 0.1% (v/v) Triton X-100, and all assays are carried out at this constant concentration of Triton X-100.

Analytical HPLC methods- Method A: The reaction products were analyzed using an

Agilent 1200 Series HPLC system and a Zorbax Eclipse XDB-C18 LC-Column (4.6 x 50 mm, 1.8 μm, Agilent) with a Zorbax Eclipse XDB-C18 guard column (4.6 x 12.5 mm, 5

μm, Agilent) or a Nova-Pak C18 reverse-phase analytical column (3.9 x 150 mm, 4 μm,

Waters) with a Nova-Pak C18 guard column (3.9 x 20 mm, 4 μm, Waters). The column

33 thermostat was set to 35°C and the flow rate was 1 mL/min. The following elution profile was used with 75:25 (v/v) acetonitrile-water with 0.1% NH4CH3COO (solvent A) and

72:18:10 (v/v) isopropyl alcohol-acetonitrile-water with 0.1% NH4CH3COO (solvent B): gradient from 100% solvent A-100% solvent B over 10 minutes, 100% solvent B for 10 minutes, gradient from 100 solvent B-100% solvent A over 6 s, and 100% solvent A for 4 minutes. Elution was monitored at 380, 414 and 453 nm.

Method B: Retinal isomers were separated on a YMC Carotenoid S-3 column (4.6 x 250 mm, 3 μm). The following elution profile was used with water (solvent A) and 75:25

(v/v) acetonitrile-methyl tert-butyl ether (solvent B): 55%B for the first five minutes, followed by a gradient from 55%B-62.5 %B over 25 minutes. The flow rate used was 1 mL/min and the column thermostat was set to 35°C. Elution was monitored at 380 nm.

Method C: β-Carotene isomers were analyzed using a YMC-Carotenoid S-3 column (4.6 x 250 mm, 3 μm), with 75:25 (v/v) methanol- MTBE as mobile phase, a flow rate of 1.4 mL/min and a column temperature of 35°C. This was based on the HPLC method of

Maeda et al. (132) Elution was monitored at 450 nm.

Method D: For multiple reaction monitoring (MRM) analysis, the BCO1-lycopene reaction mixture was separated by an Agilent 1200 SL HPLC system (Agilent

Technologies) using a YMC C30 column with dimensions 150 mm × 4.6 mm, 5 μm

(Waters). The flow rate was 1.3 mL/min, and the column temperature was 35 °C. The

34 composition of solvents was as follows: A = 80:20: methanol/water with 0.04% (w/v) ammonium acetate; B = 78:20:2 MTBE/methanol/water with 0.04% (w/v) ammonium acetate. A linear eluting gradient was applied as follows: 0-35.6% B for over 9 minutes,

35.6-100% B over the 6.5 minutes, isocratic 100%B for 2.5 minutes, 100%B-100%A over 0.1 min, and re-equilibrated for 3.4 minutes.

HPLC-MS/MS method for confirmation of acycloretinal produced from the reaction of

BCO1 and lycopene- The BCO1-lycopene reaction mixture was separated using the

HPLC method D described in the previous section. The HPLC was interfaced with a

QTrap 5500 mass spectrometer (ABSciex) operated as a triple-quadrupole using an atmospheric pressure chemical ionization (APcI) source in positive ion mode. Additional settings were as follows: nebulizer current, 5 μA; nebulizer temperature, 500 °C; declustering potential, 80 V. MS/MS transitions were optimized for acycloretinal using authentic standard prepared by organic synthesis. The MRM sequence included transitions m/z 285>69, 119, 135, and 161 using collision energies of 28, 15, 15, and 14 eV, respectively.

Kinetic data- Data from plots of reaction velocity vs. substrate concentration were fit to the Michaelis-Menten equation using GraphPad Prism 4.

2.3 Results

Expression and purification of recombinant human BCO1- The pET-28b-human BCO1 plasmid was sequenced to verify the alignment with the sequence reported in PubMed

(accession number NM_017429.2, corresponding to a 547-amino acid peptide). The clone has an additional 8 amino acids at the C-terminus corresponding to the hexahistidine tag. The theoretical mass of the protein including the hexahistidine tag is

63,702 Da.

The protein tends to form inclusion bodies under conditions of that favor high rate of expression (high IPTG and high growth temperature). Five liters of culture grown under the conditions described above yields 2-3 mg of purified protein. Sample purification gels are shown in Figure 2.1. The enzyme is soluble in detergent-free phosphate buffer. However we found greater activity if detergent is maintained through purification and storage. The average specific activity of our enzyme preparation is 132.1

± 23.8 (mean ± SD, n = 5) nmol retinal/mg BCO1/h, measured using 20 μM β-carotene and 500 ng BCO1 per reaction. Our BCO1 preparation is active even without the addition of Fe2+, and all enzyme incubations are performed without addition of the latter.

The kinetic data for β-carotene is shown in Figure 2.3. The production of retinal is linear up to 15 minutes and 500 ng of enzyme/200 μL reaction (Figure 2.2, A and B).

Purified recombinant human BCO1 cleaves β-carotene solely at the central bond- We found trace amounts of β-apocarotenals in the β-carotene-BCO1 reaction mixtures. We first conducted experiments to see if BCO1 catalyzed cleavage of β-carotene at positions

36 other than the 15-15′ bond. However, there was no evidence of enzyme-dependent formation of these β-apocarotenals (Figure 2.3). Even at the higher protein concentrations tested when making the BCO1 protein curve (Figure 2.2B), there was still no evidence that β-apocarotenals are formed enzymatically. Thus, BCO1 cleaves solely at the central

15-15′ bond.

Purified recombinant BCO1 cleaves β-apocarotenals to yield retinal and lycopene to yield acycloretinal- Six major dietary carotenoids (β-carotene, lycopene, α-carotene, β- cryptoxanthin, zeaxanthin, lutein) as well as β-apocarotenals (β-apo-8′-carotenal, β-apo-

10′-carotenal, β-apo-12′-carotenal and β-apo-14′-carotenal) were tested for activity with

BCO1, comparing peak areas from 15-minute reactions with time-zero, boiled enzyme and no enzyme controls. We detected enzymatic cleavage activity with all of the test substrates except for zeaxanthin and lutein. For the asymmetrical provitamin A carotenoids α-carotene and β-cryptoxanthin, two product peaks were detected, one corresponding to all-trans-retinal, and the other corresponding to the non-retinal half of the molecule. However, we only quantified the amount of all-trans-retinal produced in the reaction. For lycopene, acycloretinal was detected as product (Figure 2.4), and synthetic acycloretinal was used to generate standard curves for quantification.

Time courses were run for all other substrates using 500 ng BCO1/200 μL reaction, and the production of retinal (or acyloretinal in the case of lycopene) was linear within the first 15-25 minutes (data not shown). The dependence of reaction velocity on substrate concentration is shown in Figures 2.2C, 5 and 6. The carotenoids and β-

37 apocarotenals vary in solubility and this affects the range of concentrations that can be tested.

The kinetic data for all substrates tested and their values relative to β-carotene are shown in Figure 2.7. With the exception of β-apo-8′-carotenal, the substrate curves for the β-apocarotenals do not fit well with the Michaelis-Menten equation (Figure 2.6).

The kinetic data with the full-length carotenoids suggest that a β-ionone ring conjugated to the rest of the polyene chain is essential for activity. Hydroxylation of the ring (as in β-cryptoxanthin) or loss of conjugation between the ring and the rest of the polyene chain (as in α-carotene) result in a decrease in catalytic activity. Hydroxylation of both rings (as in zeaxanthin and lutein) results in a loss of activity. Surprisingly, lycopene is cleaved by BCO1 at a catalytic efficiency similar to β-carotene. Although the

Vmax of lycopene is low, the KM is also low, thus resulting in a relatively high kcat/KM value.

Purified recombinant human BCO1 does not cleave 9-cis-β-carotene- Figure 2.8A-C shows the chromatograms for the Betatene ® hexane extract, all-trans-β-carotene standard, and the purified 9-cis-β-carotene. We incubated 9-cis-β-carotene (20 μM) with

BCO1 under standard conditions (500 ng/200 μL reaction, 15 minutes reaction time), but did not detect any difference between the amount of retinals in the 15-minute reaction and time-zero control (data not shown). We then incubated 9-cis-β-carotene (15 μM) with

2500 ng BCO1 and 60 minutes reaction time (Figure 2.8D). There were trace amounts of

9-cis-and all-trans-retinal detected in the reaction mixture, but their amounts are the same

38 as those detected in the controls (time-zero, no enzyme, heat-denatured enzyme).

Incubation of all-trans-β-carotene under the same conditions yields a significant amount of retinal compared to the same controls (Figure 2.8E). Thus, purified recombinant human BCO1 does not cleave 9-cis-β-carotene.

2.4 Discussion

Eccentric cleavage products of β-carotene (β-apocarotenoids) have been detected in foods such as melons (147) and peppers (148), and in human plasma (149). They have also been shown to have an effect of cell differentiation and proliferation (150-151), and to modulate the activity of nuclear receptors (68-69,152). Thus, there is great interest in their origins and metabolism. Carotenoids are susceptible to non-enzymatic oxidation that yields β-apocarotenoids, but the assay system that we adapted from During et al.

(68,142) has two features to avoid this. First, an antioxidant, α-tocopherol, is used, and this has been shown to reduce the occurrence of eccentric cleavage products in incubations of β-carotene with tissue homogenates (114). Second, the assay is much shorter because there is no liquid-liquid extraction of reaction products, evaporation of solvent and reconstitution with an organic solvent. A mixture of acetonitrile and isopropyl alcohol is added to the aqueous reaction mixture, and the resulting solution is filtered and injected into the HPLC. This makes processing even faster than the original method, which calls for centrifugation for 10 minutes after addition of organic solvent.

Filtration is made easier because of the much lower amounts of purified enzyme used

(250-2500 ng) compared to the amounts of protein used in tissue homogenate incubations 39

(0.5-1 mg) (142,145). Our HPLC method was also able to separate the four β- apocarotenals that could be potentially produced by eccentric cleavage (Figure 2.3).

Thus, we easily identified the peaks in our chromatograms, compared the amounts present in the reaction mixture with three controls (time-zero, no enzyme and heat- denatured enzyme), and determined that purified recombinant human BCO1 does not produce β-apocarotenals, even with increased enzyme concentrations.

We also show that the β-apocarotenals of 22 carbons or greater are substrates for

BCO1. The β-apocarotenals are more water-soluble than full length carotenoids, which is why we were able to test higher concentrations for constructing substrate-reaction velocity curves. However, we did not determine the loading capacity of the Tween-40 micelles used in the assay. It is possible that at higher concentrations, some of the β- apocarotenal substrate molecules are in micelles while the others are dissolved in water, and thus introduces variability in the way the substrate is presented to the enzyme. This could explain why the substrate-reaction velocity curves for β-apocarotenals 27 carbons or shorter do not fit well with the Michaelis-Menten equation (Figure 2.6).

The experiments reported here show that purified recombinant human BCO1 does not cleave 9-cis-β-carotene. Again, the shorter processing time of our assay and the use of amber lights minimized non-enzymatic isomerization, and the use of purified protein and three controls make the interpretation of results straightforward. We expected some activity based on previous studies where 9-cis-β-carotene was incubated with supernatants of rat liver and intestine homogenates (145), and the extract from E.coli expressing recombinant murine BCO1 (132). Also, the incubation of 9-cis-β-carotene

40 with the post-nuclear fraction from human intestinal mucosa was reported to yield 9-cis- retinoic acid, suggesting that 9-cis-β-carotene was cleaved, producing 9-cis-retinal which was then oxidized to the acid (146). It is possible that, since we are using purified enzyme, we are missing cofactors/coenzymes present in crude enzyme preparations, and we cannot discount the possibility that BCO1 might still cleave 9-cis-β-carotene in vivo.

On the other hand, results with tissue homogenates should also be interpreted with caution, as the substrate might be acted on by enzymes other than BCO1.

It has been reported that intraperitoneal administration of 9-cis-β-carotene does not increase the amount of 9-cis-retinoids measured in the eyes and livers of WT and

BCO1 KO mice (132). Several human studies have shown that oral 9-cis-β-carotene is absorbed poorly or not at all (15,17,144,153). These studies suggest that 9-cis-β-carotene is poorly metabolized by humans, and our results are consistent with this picture. This calls into question the value of supplementation with Betatene® or other similar

Dunaliella extracts, especially in the context of vitamin A supplementation. Furthermore, the inactivity of 9-cis-β-carotene with human BCO1 also suggests that the increase of cis isomers of β-carotene during food processing decreases its provitamin A value.

The discovery of 9-cis-retinoic acid as a ligand for retinoid X receptors (RXR’s)

(154-155) has, of course, led to the question of its biosynthesis. The central cleavage of

9-cis-β-carotene to produce 9-cis-retinal and subsequent oxidation to 9-cis-retinoic acid has been proposed as a possible mechanism (156). However, our results argue against this, and it is more likely that the all-trans-retinoic acid/retinol that originated from all- trans-β-carotene is isomerized to the 9-cis-form. It has been reported that all-trans-

41 retinoic acid can be isomerized to 9-cis-retinoic acid by thiolate radicals (157-158).

Another study suggests that an isomerase converts all-trans-retinol to 9-cis-retinol (159).

Our results with the major dietary carotenoids are largely in agreement with other in vitro studies that used recombinant BCO1, with the notable exceptions of our detection of activity with lycopene and β-apocarotenoids shorter than β-apo-10′-carotenal (93,117).

During our initial attempts to generate a lycopene substrate curve, we used the same concentration range as β-carotene (2.5-20 μM). We observed the highest activity for the lowest substrate concentration, and above 10 μM, the substrate solutions were cloudy, meaning lycopene was not successfully micellarized. Thus, we decided to expand the low concentration range from 0.3-2.5 μM, and generated a substrate curve that shows maximum activity at 2.2 μM. It is possible that other in vitro studies were not able to detect activity with lycopene because they used relatively high concentrations that favored precipitation. We also found that exposure of the substrate solution to low temperatures at any point during the substrate preparation leads to a cloudy mixture.

Examples of such conditions include evaporative cooling during removal of the organic solvent, and storage of the substrate solution in ice. Thus, in order to maximize activity as well as minimize non-enzymatic isomerization and oxidation, the substrate should be prepared just before use, and be kept no lower than room temperature (about 22°C) until used.

The cleavage of lycopene to produce acycloretinal suggests that it is possible to obtain acycloretinol and/or acycloretinoic acid in vivo. Acycloretinoic acid has been shown to induce apoptosis in human prostate cancer cell lines (160), activate the DR-5

42 retinoic acid response element in MCF-7 cells (161), and stimulate gap junction communication (162). However, these studies show that supraphysiological concentrations are necessary for activity (160), or that acycloretinoic acid is less effective than retinoic acid (161) or lycopene (162).

However, acycloretinal and its alcohol and acid forms have not been detected in vivo (163-166). It is possible that very small amounts of acycloretinal are generated in vivo because of the low solubility of lycopene, an echo of our experience with the in vitro assays. Furthermore, the substrate that we used is 92% all-trans-lycopene, whereas a significant proportion (25-70%) of the lycopene in human body fluids and tissues are various cis isomers (12). Human BCO1 might cleave these cis isomers less efficiently or not at all.

Our results with lycopene show that the presence of an unmodified β-ionone ring is not crucial for activity with BCO1. For β-carotene and lycopene, there is a conjugated system starting from the 5-carbon to the 5′-carbon. In α-carotene, which is cleaved less efficiently than β-carotene and lycopene, the conjugated system is disrupted. Apparently, it is the structure of conjugated system rather than the ring itself that is important for activity of BCO1 with the molecule. As of the time of writing, there is no crystal structure available for BCO1. There is only one structure of a retinal-forming carotenoid oxygenase known, that from the cyanobacterium Synechocystis sp. PCC 6803 (83). In this structure, the pocket where the carotenoid is held is lined with numerous nonpolar residues, mainly with aromatic side chains. It is thus possible that the conjugated system in the carotenoid forms π-π interactions with these side chains, and an alteration of the

43 conjugated system, as in α-carotene, interferes with the binding. This is supported by the higher KM observed for α-carotene. Also, the α-ionone ring is free to rotate about the 6-7 bond, unlike the β-ionone ring. The loss of the planarity in this part of the molecule may also contribute to the lower binding observed. Hydroxylation of the ring significantly also affects the activity of BCO1 with the molecule, as shown by the lower activity with β- cryptoxanthin, and lack of activity with zeaxanthin and lutein. This might be due to unfavorable interaction of the hydroxyl group with the nonpolar residues in the substrate binding pocket.

As shown by this work and earlier studies, β-carotene is still the most effective provitamin A carotenoid in the context of BCO1 cleavage. However, there are other factors that will affect the effectiveness of provitamin A carotenoid to be converted into vitamin A in vivo. The molecular structure is only one of the many factors that influence the bioavailability and bioconversion of carotenoids (reviewed by Castenmiller et al.)

(3,167). Nevertheless, an in vitro substrate specificity study using purified enzyme is an important component of interpreting what is observed in vivo.

In summary, human BCO1 is, with the notable exception of its ability to cleave lycopene, mostly a provitamin A carotenoid oxygenase. It catalyzes the oxidative cleavage of major dietary provitamin A carotenoids and β-apocarotenals solely at the 15-

15′ bond to produce retinal. Lutein and zeaxanthin, which will not yield retinal with central cleavage, do not react with BCO1. BCO1 does not catalyze the cleavage of 9-cis-

β-carotene. The production of acycloretinal from lycopene by BCO1 is contrary to the

44 majority of previous reports. This warrants a fresh look at acycloretinal and its alcohol and acid forms as metabolites of lycopene in future studies.

2.5 Figures

Figure 2.1. Purification of recombinant human BCO1 using cobalt affinity column chromatography. The SDS-PAGE gel on the left shows the elution of recombinant His- tagged human BCO1 (theoretical MW=63,702 Da) from the cobalt column by increasing concentrations of imidazole. Only the fractions that show a single band were combined and desalted into the final storage buffer (lanes 5-8). The SDS-PAGE gel on the right shows the combined fractions and the protein obtained after desalting.

Figure 2.2. Kinetic data for purified recombinant BCO1 with β-carotene at 37°C. Each data point represents the difference between the average of duplicate measurements at a given reaction time and the average of duplicate time-zero controls. A) Time course using 5 μM β-carotene incubated with 250 ng BCO1. B) Protein curve using 20 μM of β- carotene and 15 minutes reaction time. C) Plot of reaction velocity as a function of β- carotene concentration using 500 ng BCO1/200 μL reaction and 15 minutes reaction time. The red trace is the best-fitting Michaelis-Menten curve generated by GraphPad Prism 4. Three independent substrate-velocity plots were generated to calculate the average kinetic parameters stated in Figure 2.7.

Figure 2.3. Purified recombinant BCO1 cleaves β-carotene solely at the central 15- 15′ bond. A) Chromatograms from the reaction mixture of β-carotene (20 μM) and BCO1 (500 ng/200 μL reaction) at 37°C. Blue trace-15 min reaction, red trace-time-zero control. The product peak has the same retention time and UV-Vis spectrum as standard all-trans-retinal. The following chromatograms are for standards B) all-trans-retinal, C) β-apo-14′-carotenal, D) β -apo-12′-carotenal, E) β -apo-10′-carotenal, and F) β-apo-8′- carotenal. Chromatography was performed using Zorbax Eclipse XDB-C18 LC-Column (4.6 x 50 mm, 1.8 μm, Agilent) and HPLC Method A described under Materials and Methods. Signal monitored: 380 nm.

Figure 2.4. Purified recombinant BCO1 cleaves lycopene to produce acycloretinal. A) Sample chromatograms for reaction mixtures of BCO1 (200 μL reaction) with lycopene (2.2 μM) at 37°C. The chromatograms shown are for the reactions with 2500 ng BCO1 at 15 min (blue), 500 ng BCO1 at 15 min (violet), 500 ng BCO1 at time zero (red), no enzyme at 15 minutes (orange) and 500 ng heat-denatured BCO1 at 15 minutes (green). (B) Chromatogram for acycloretinal standard. Chromatography was performed using Zorbax Eclipse XDB-C18 LC-Column (4.6 x 50 mm, 1.8 μm, Agilent) and HPLC Method A described under Materials and Methods. Signal monitored: 380 nm. The acycloretinal product of the reaction of lycopene and BCO1 was confirmed by multiple reaction monitoring (MRM). MRM chromatograms are shown for the reaction of 2.2 μM lycopene with 2500 ng BCO1/200 μL at 15 min and 37°C (C), and the acycloretinal standard (D). The chromatography was performed using HPLC Method D, and MS/MS conditions are described under Materials and Methods. Four transitions were monitored– m/z 285>69 (blue), 119 (red) and 135 (green) and 161 (grey). The matching retention times and relative intensities of the transitions confirm the identity of the product as acycloretinal.

Figure 2.5. Substrate-velocity plots for other substrates that display Michaelis- Menten behavior. The reactions were performed using 500 ng BCO1/200 μL reaction and 15 minutes reaction time at 37°C. Each data point represents the difference between the average of duplicate measurements at a given reaction time and the average of duplicate time-zero controls. The red traces are the best-fitting Michaelis-Menten curves generated by GraphPad Prism 4. Three independent substrate-velocity plots were generated to calculate the kinetic parameters stated in Figure 2.7.

Figure 2.6. Substrate-velocity plots for β-apocarotenals. The reactions were performed using 500 ng BCO1/200 μL reaction and 15 minutes reaction time at 37°C. Each data point represents the average of three independent experiments. Only β-apo-8′-carotenal displays Michaelis-Menten kinetics (shown in Figure 2.5) in the assay conditions used.

Figure 2.7. Kinetic data for purified recombinant BCO1 and various carotenoids and apocarotenals. Results are calculated from three independent experiments. The Vmax and KM values shown are the means ± SD.

Figure 2.8. Purified recombinant human BCO1 does not cleave 9-cis-β-carotene. Chromatograms for A) Betatene® extract obtained from liquid-liquid extraction with the β-carotene (β-C) isomer peaks indicated, B) all-trans-β-carotene standard, C) 9-cis-β- carotene obtained using HPLC Method C described under Materials and Methods. Signal monitored: 450 nm. The test carotenoids (15 μM) were incubated with 2500 ng purified recombinant human BCO1 for 1 hour, and analyzed using HPLC Method B described under Materials and Methods. No enzyme-dependent production of retinal was observed for 9-cis-β-carotene (D), in contrast to all-trans-β-carotene (E).

Chapter 3: Substrate specificity of purified recombinant chicken BCO2

ABSTRACT

Provitamin A carotenoids are oxidatively cleaved by β-carotene 15-15′-oxygenase

(BCO1) at the central 15-15′ double bond to form retinal (vitamin A aldehyde). Another carotenoid oxygenase, β-carotene 9′-10′-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9′-10′ bond to yield an ionone and an apo-10′-carotenoid.

Previously published substrate specificity studies of BCO2 have been conducted using crude lysates from bacteria or insect cells expressing recombinant BCO2. Our attempts to obtain active recombinant human BCO2 expressed in E.coli were unsuccessful. We have expressed recombinant chicken BCO2 in the E.coli strain BL21-Gold (DE3) and purified the enzyme by cobalt ion affinity chromatography. Like BCO1, purified recombinant chicken BCO2 reacts with the provitamin A carotenoids β-carotene, α-carotene, and β- cryptoxanthin. In contrast to BCO1, purified recombinant chicken BCO2 also reacts with the nonprovitamin A carotenoids zeaxanthin and lutein, and does not react with all-trans- lycopene and β-apocarotenoids. Apo-10′-carotenoids were detected as enzymatic products by HPLC, and the identities were confirmed by LC-MS. Small amounts of 3- hydroxy-β-apo-8′-carotenal were also consistently detected in BCO2-β-cryptoxanthin reaction mixtures. With the exception of this activity with β-cryptoxanthin, BCO2 cleaves specifically at the 9′-10′ bond to produce apo-10′-carotenoids.

3.1 Introduction

There are three members of the carotenoid oxygenase family expressed in vertebrates. β-Carotene 15-15′-oxygenase (BCO1) reacts with provitamin A carotenoids to form retinal (vitamin A aldehyde). RPE65 is an isomerohydrolase that reacts with all- trans-retinyl esters to form 11-cis-retinol, and is an important component of the visual cycle (74-78). β-Carotene 9′-10′-oxygenase (BCO2) catalyzes the oxidative cleavage of carotenoids at the 9′-10′ bond to yield an ionone and an apo-10′-carotenoid. Studies in mice suggest that BCO2 cleavage prevents oxidative stress from carotenoid accumulation, especially in the mitochondria (126). The provitamin A carotenoids β- carotene and β-cryptoxanthin are also cleaved by BCO2 to produce β-apo-10′-carotenal

(126,130,168), which can then be oxidatively cleaved by BCO1 to produce retinal (168-

169).

Unlike BCO1, BCO2 has also been shown to cleave non-provitamin A carotenoids such as zeaxanthin and lutein to produce 3-hydroxy-apo-10′-carotenals

(126,130). The significance of these compounds in mammals is unknown. However,

(3R)- hydroxy-β-apo-10′-carotenol, also known as galloxanthin, has been identified as one of the dominant carotenoids in quail retina (170). This is presumably formed from the reduction of (3R)-hydroxy-β-apo-10′-carotenal, which can be formed from BCO2 cleavage of either zeaxanthin or lutein (Figure 3.1). Ferret hepatic homogenates have been shown to reduce of (3R)-hydroxy-β-apo-10′-carotenal to the alcohol (130) The production of β-apo-10′-carotenol has been shown in vivo. BCO1 KO mice, which have elevated expression of BCO2, accumulate β-apo-10′-carotenol on a diet with only β-

56 carotene as the sole source of apocarotenoids (171). In humans, the dominant carotenoids in the retina are lutein, 3′-epilutein and zeaxanthin(170). Given that BCO2 is also highly expressed in the human retinal epithelium (131), it is interesting that BCO2 cleavage products of xanthophylls are not the dominant carotenoids. It is possible that human

BCO2 and avian BCO2 have different substrate specificities, or another regulatory mechanism(s) exist to favor the BCO2 cleavage of lutein and zeaxanthin in avian retina but not in humans.

The published BCO2 substrate specificity studies as of the time of writing have been done with crude lysates of bacteria or insects cells expressing recombinant BCO2

(126,130). In this chapter, we describe the substrate specificity of purified recombinant chicken BCO2. We show that the enzyme reacts with β-carotene, α-carotene, β- cryptoxanthin, zeaxanthin and lutein, but not with lycopene and β-apocarotenals.

3.2 Materials and Methods

Carotenoids and retinoids- β-Carotene (≥97%), all-trans-retinal (≥98%), 9-cis-retinal

(≥95%), 13-cis-retinal (≥85%), α-carotene (≥95%), β-cryptoxanthin (≥97%), zeaxanthin

(≥95%), lutein (≥90%) and β-apo-8′-carotenal (≥96%) were purchased from Sigma-

Aldrich. The lycopene standard (92% all-trans, 6% 5-cis, 2% other isomers) was a gift from Dr. Steven J. Schwartz of The Ohio State University. β-Apo-10′-carotenal, β-apo-

12′-carotenal and β-apo-14′-carotenal were synthesized according to published methods

(68).

Plasmids- Plasmids for bacterial expression containing human BCO2 isoforms a and d with a C-terminal His-tag were purchased from Genecopoeia. A pET-28a plasmid vector containing the cDNA of chicken BCO2 with an N-terminal hexahistidine tag was a gift from Dr. Matthew B. Toomey of Washington University at St. Louis.

Expression and purification of recombinant BCO2- For human BCO2 isoforms a and d, the plasmid was transformed into E.coli BL21-Gold (DE3) (Stratagene) or T7 Express

(New England Biotechnologies) according to the manufacturer’s instructions. For chicken BCO2, the plasmid was transformed into E.coli BL21-Gold (DE3). The transformed bacterial cells were grown in LB broth (Sigma-Aldrich) at 30°C to an OD600 of 0.5-0.7. The incubator temperature was then lowered to 16°C and expression of the recombinant protein was induced by adding IPTG (Gold Biotechnology) to a final concentration of 0.1 mM. Five liters of culture were grown for 16 hours and the cells were harvested by centrifugation. The cell pastes were frozen at -80°C and thawed prior to lysis. The protein was purified using the same protocol for recombinant human BCO1

(169).

In vitro BCO2 assay- The in vitro BCO1 enzyme assay was based on the method of

Amengual et al. (126). All substrates were tested at a concentration of 20 μM at 37°C with the indicated incubation time and protein concentration. The working enzyme solution for a single reaction is prepared by combining the required amount of enzyme and storage buffer to a total volume of 100 μL, 40 μL of reaction buffer (250 mM

Tricine-KOH, pH 8.0 at 37°C, 625 mM NaCl), 4 μL of 500 μM FeSO4, 2 μL of 0.5 M tris

(2-carboxyethyl)phosphine (TCEP) and 14 μL of deionized water. The substrate solution was prepared by first mixing 18 nmol of carotenoid in hexanes or ethanol, 9 μL of 10 mM α-tocopherol, 225 μL of 4% octylthioglucoside in ethanol and 400 μL of acetone.

The mixture was dried under nitrogen, re-dissolved in 200 μL of acetone, dried down again, and re-dissolved in 180 μL of deionized water. The substrate and enzyme solutions were incubated at 37°C for 5 minutes, and the reaction was initiated by adding 40 μL of the substrate to the enzyme, for a total reaction volume of 200 μL. The reaction was quenched with 50 μL of 37% formaldehyde and incubated for a further 10 minutes at

37°C. Then, 500 μL of 1:1 (v/v) acetonitrile-isopropyl alcohol was added after quenching the reaction with formaldehyde. The resulting mixture was filtered through a 0.22 μM syringe filter and 100 μL was subjected to HPLC. Time zero, no enzyme and heat- denatured enzyme controls were used.

Analytical HPLC methods- The reaction products were analyzed according to HPLC

Method A described in section 2.2.

For lutein, the chromatography described above cannot separate the 3-hydroxy-β- apo-10′-carotenal and 3-hydroxy-α-apo-10′-carotenal produced from its reaction with

BCO2. However, these were separated by the following chromatography system: column,

Zorbax XDB-C18 (Waters), 4.6 x 150 mm, 5 μm particle size; flow rate, 1.5 mL/min; column temperature, 35°C. The following elution profile was used with 80:20 (v/v) methanol-water with 0.1% NH4CH3COO (solvent A) and 78:20:2 (v/v) MTBE-methanol

59 water with 0.1% NH4CH3COO (solvent B): gradient from 0-40% solvent B over 8 minutes, gradient from 40-95% solvent B over 1 minute, gradient from 95-100% solvent

B over 1 minute, and gradient from 100-0%B over 4 minutes.

LC-MS analysis- HPLC was performed HPLC Method A described in section 2.2 with the following modifications. First, formic acid was used instead of ammonium acetate.

Second, the column used was also Zorbax XDB-C18, but with dimensions of 4.6 x 150 mm, and 5 μm particle size.

The HPLC was interfaced with a quadrupole time-of-flight (Q-TOF) mass spectrometer (Q-TOF Premier, Micromass, UK) via an APcI probe. β-Carotene, β-apo-8′- carotenal, β-apo-10′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal, and retinal were ionized in the APcI negative mode as their respective radical anions, 536.438, 416.310,

376.280, 350.260, 310.230, and 284.210 m/z. The Q-TOF system allowed for quantitative detection with the confidence of accurate mass typically being 1 ppm. Mass spectra were acquired in V-mode (∼8000 resolution) from 50–1000 m/z with a scan time of 1 s, peak centroiding, and enhanced duty cycle enabled for the parent m/z. At intervals of 30 s, a

0.1 s lockspray scan was acquired with leucine enkephalin as the lockspray compound

(554.2615 m/z) to correct for minor deviations in calibration due to temperature fluctuations. Prior to analysis, the Q-TOF was fully calibrated from 114 to 1473 m/z using a solution of sodium formate. The resultant MS spectra were acquired and integrated with MassLynx software, V4.1 (Micromass UK, Manchester, UK). Source parameters were: 30 μA corona current; 550 °C probe; 110 °C source block; 35 V cone;

100 L/h cone gas (N2); 400 L/h desolvation gas (N2); and collision energy 8 eV (non- fragmenting) with argon as the CID gas (4.2 × 10−3 mBar).

3.3 Results

Recombinant human BCO2 expressed in E.coli is inactive- As of the time of writing, there are six isoforms of human BCO2 identified in Pubmed (172). The longest is isoform a (NM_031938.5), corresponding to a 579-amino acid peptide (Figure 3.2).

Isoform d (NM_001256398.1) corresponds to a 506-amino acid peptide and is the same sequence used by Kim et al. (129) in their paper claiming to produce active recombinant human BCO2 in E.coli. strain ER2566 (equivalent to T7 Express).

Both isoforms behave similarly to human BCO1 (Chapter 2)-high rates of expression favor formation of inclusion bodies, but the small amount of His-tagged protein can be recovered from the supernatant by cobalt ion affinity chromatography.

However, there is no detectable activity from the crude supernatant and the purified protein from either strain of E.coli tested. Furthermore, we did not detect any activity when the in vitro assay was conducted in the same manner as BCO1 (Chapter 2) or by the method of Amengual et al.(126).

Expression and purification of recombinant chicken BCO2- The pET28a-chicken BCO2 clone is similar to the isoform x5 of Gallus gallus BCO2 mRNA in PubMed (accession number XM_417929.4, corresponding to a 579-amino acid peptide), but the first 31

61 amino acids of the latter are replaced with 16 amino acids corresponding to the His tag

(Figure 3.3).

As with recombinant human BCO2 and BCO1, recombinant chicken BCO2 is not very soluble under the expression conditions in BL21-Gold (DE3), but the small amount of soluble protein was easily recovered to a high degree of purity by cobalt ion affinity chromatography.

Chicken BCO2 cleaves full-length β-ring carotenoids at the 9′-10′ bond- The activity of purified recombinant chicken BCO2 was tested with the major dietary carotenoids (β- carotene, α-carotene, β-cryptoxanthin, zeaxanthin, lutein, lycopene) and β-apocarotenals

(β-apo-8′-carotenal, β-apo-10′-carotenal, β-apo-12′-carotenal and β-apo-14′-carotenal), using 20 μM of each substrate, with time-zero, no-enzyme and heat-denatured enzyme controls. Only full-length carotenoids with at least one β-ring (β-carotene, α-carotene, β- cryptoxanthin, zeaxanthin, lutein) were cleaved (Figures 3.4-3.6, 3.8 and 3.9) to the corresponding apo-10′-carotenal. Also, an increase in the amount of protein shows an increase in the amount of the apo-10′-carotenal product(s), but we did not see any evidence of cleavage at other positions, except with β-cryptoxanthin (Figure 3.6). 3-

Hydroxy-β-apo-8′-carotenal, a product of 7-8 double bond cleavage, was identified by

LC-MS and consistently observed above the level of controls (peak 2 in Figure 3.6). The

UV-Vis spectra of the peak is also consistent with 3-hydroxy-β-apo-8′-carotenal. The chromophore of the carotenoids and apocarotenoids is the conjugated double bond system, and hydroxylation that does not disrupt the latter does not cause large shifts in

λmax (173). Thus, the UV spectra of 3-hydroxy-β-apo-10′-carotenal and β-apo-10′- carotenal show the same λmax (Figure 3.7) However, the UV spectrum of peak 2 is more similar to that of standard β-apo-8′-carotenal rather than β-apo-10′-carotenal.

It has been reported that murine BCO2 can cleave the apo-10′-carotenal product the yield rosafluene dialdehyde (126) (Figure 3.9). However, by identification of the masses of the cleavage products by LC-MS, we did not find evidence of its formation even with increase in reaction time (up to 2 hours) and 5 μg protein/200 μL reaction.

Also, incubation of β-apo-10′-carotenal with chicken BCO2 did not show any evidence of a reaction.

3.4 Discussion

As reviewed in section 1.3, products of excentric cleavage of carotenoids are important in the production of plant hormones such as abscisic acid and strigolactone. For vertebrates, the central cleavage product of provitamin A carotenoids, retinal, is still the most important in terms of function. Our finding that the central bond cleavage enzyme

BCO1 cleaves only provitamin A carotenoids (except lycopene) specifically at the 15-15′ bond to yield retinal is only consistent with the latter’s biological importance. Thus, the existence of a 9′-10′ bond oxygenase in vertebrates is interesting. Current literature suggests that the primary function of BCO2 in mice is to prevent oxidative stress from accumulation of carotenoids in the mitochondria (126), and so far, a biological activity for β-apo-10′-carotenoids has not been identified (apart from their provitamin A activity)

(174). 63

Our results show that, with the exception of β-cryptoxanthin, chicken BCO2 cleaves carotenoids specifically at the 9′-10′ position to yield an apo-10′-carotenal. The ability of chicken BCO2 to cleave β-cryptoxanthin at the 7′-8′ to produce 3-hydroxy-β- apo-8′-carotenal is possibly due to the asymmetry of the molecule, causing a minor, alternative binding mode with BCO2. Our experiments with BCO1 show that hydroxylation of the ring in β-cryptoxanthin results in lower KM compared to β-carotene, and total loss of activity when the two rings are hydroxylated as in zeaxanthin (Figure

2.8). This suggests that a hydroxyl group may form unfavorable interactions with the hydrophobic substrate tunnel of BCO1, or is simply an issue of size i.e. the 3-hydroxyl β- ionone ring is too large to pass through the substrate tunnel. The ability of BCO2 to cleave both carotenoids with and without 3-hydroxyl groups on the ionone rings, as shown by this study and others (126,130), suggests that the substrate tunnel of BCO2 is larger than BCO1, and/or the 3-hydroxyl groups do not form unfavorable interactions with the hydrophobic substrate tunnel. Kinetic data obtained using lysates from ferret

BCO2-expressing Sf9 cells also suggest that lutein and zeaxanthin, which both contain 3- and 3′-hydroxyl groups, are better substrates than β-cryptoxanthin, which contains only one 3-hydroxyl group (130). Our semi-quantitative results, at this point, also support this, although determination of Michaelis-Menten kinetic parameters is necessary to make this conclusive.

The relatively large difference in peak areas of the product peaks from the asymmetrical carotenoid substrates (α-carotene, β-cryptoxanthin and lutein) (Figure 3.5,

3.6 and 3.8) suggests that the enzyme displays an apparent regioselectivity. With β-

64 cryptoxanthin, the production of β-apo-10′-carotenal is favored over the alternative cleavage product. With α-carotene, α-apo-10′-carotenal is the favored cleavage product, i.e. the cleavage of the 9-10 bond closer to the β-ionone ring is favored. On the other hand, with lutein, which is the 3,3′-dihydroxy form of α-carotene, the favored cleavage product is 3-hydroxy-β-apo-10′-carotenal, i.e. the cleavage of the 9-10 bond closer to the

α-ionone ring is favored. Xanthophylls such as lutein are more abundant in the diet of birds compared to their hydrocarbon counterparts (175). Thus, it is not surprising that chicken BCO2 favors the cleavage of lutein at the double bond that will yield the precursor to galloxanthin.

Unlike murine BCO2 (126), chicken BCO2 cannot perform a second cleavage on

β-apo-10′-carotenal to yield rosafluene dialdehyde. If galloxanthin is an important carotenoid in avian vision and is indeed generated via BCO2 cleavage of zeaxanthin or lutein (Figure 3.1), then it makes functional sense that chicken BCO2 does not cleave apo-10′-carotenals. If the function of BCO2 in mice is to degrade excess carotenoids, then it also makes functional sense that it can cleave full-length carotenoids twice (Figure

3.9), producing smaller molecules that are easier to clear from the circulation.

Chicken BCO2 does not react with all-trans-lycopene, which is in agreement with the report on ferret BCO2 (127). This is also consistent with the preference of BCO2 for substrates that contain a 3-hydroxy-β-ionone ring. Ferret BCO2, however, is able to cleave cis isomers of lycopene. Since the majority of lycopene in human fluids and tissues are cis isomers, then the preference of BCO2 for the latter is consistent with its proposed role of preventing excess accumulation of carotenoids. The acid, alcohol and

65 aldehyde forms of the 9′-10′ cleavage product of lycopene have shown biological activity in cell culture, inducing retinoic acid receptor β (RARβ) mRNA levels and phase II enzymes at the mRNA and protein levels (176).

Chicken BCO2 also does not react with the other β-apocarotenals in the series (β- apo-8′-carotenal, β-apo-12′-carotenal, β-apo-14′-carotenal). Again, this is consistent with the importance of the 3-hydroxy-β-ionone ring for BCO2 cleavage. β-carotene, with two

β-ionone rings, is a relatively poor substrate, and it is not surprising that the β- apocarotenals, with only one β-ionone ring per molecule, are altogether unreactive.

It is interesting that the substrates that are unreactive with BCO2 (all-trans- lycopene, β-apocarotenals) are substrates for BCO1 (Chapter 2). With the β- apocarotenals, this difference in substrate specificities of the two CCO’s is apparently to favor the production of retinal, which is not surprising given the latter’s biological importance. It is tempting to apply the same logic to all-trans-lycopene, which would imply that there is potentially some importance in favoring the production of acycloretinal. As reviewed in Chapter 2, acycloretinoids has been shown to have weak activity compared to retinoids, and they have not been detected in biological systems

(176).

In summary, our results show that chicken BCO2 has a wide substrate specificity, consistent with its proposed function of preventing oxidative stress brought about by carotenoid accumulation in the mitochondria. It cleaves only full-length carotenoids with ionone rings, and the hydroxylated carotenoids are cleaved to a greater extent than their

66 hydrocarbon counterparts under the conditions tested. Furthermore, the enzyme displays regioselectivity with asymmetrical substrates.

3.5 Table

BCO1 (142) BCO2 (126) Reaction buffer pH 8.0 8.0 Component Compound Final Compound Final concentration in concentration in reaction reaction Buffer salt Tricine-KOH 0.1 M Tricine-KOH 0.1 M Salt ----- NaCl 125 mM Reducing agent Dithiothreitol 0.5 mM TCEP 5 mM Ferrous ion ----- FeSO4 10 μM Cofactor Nicotinamide 15 mM ----- Bile salt Sodium cholate 4 mM ----- Substrate Detergent for Tween 40 0.15% Octylthio- 1% (w/v) carotenoid glucoside micelles Antioxidant α-tocopherol 0.1 mM -----*

*α-Tocopherol was not used in the original method of Amengual et al., but was used in our experiments. Exclusion of α-tocopherol from the assay had no effect.

Table 3.1. Comparison of assay components used for BCO1 and BCO2 in vitro activity assays

3.6 Figures

OH OH

HO HO

Zeaxanthin Lutein

BCO2

O HO

3-hydroxy-β-apo-10′-carotenal

Alcohol dehydrogenase

OH HO

3-hydroxy-β-apo-10′-carotenol

Figure 3.1. Production of galloxanthin from zeaxanthin or lutein by BCO2 cleavage. Zeaxanthin or lutein can yield 3-hydroxy-β-apo-10′-carotenal from BCO2 cleavage. This is then potentially reduced to galloxanthin by either retinol dehydrogenase (RDH) or by medium-chain alcohol dehydrogenases (ADH) similar to retinal (177).

Figure 3.2. Sequence alignment of the isoforms of human BCO2 using COBALT. Sequence alignment of the six isoforms of human BCO2 listed in Pubmed (172) generated by COBALT (http://www.ncbi.nlm.nih.gov/tools/cobalt/) (178). The image is generated by ESPript (179).

Tag 1 MHHHHHHSSGLVPRG------SHM------QFVP------G 23

X5 1 MPSSEQRMFSKIALSAVSILLANLRHLLSSLM------QFVP------G 37

X6 1 MPSSEQRMFSKIALSAVSILLANLRHLLSSLM------QFVPARKWVADSLAMGNLLARWNEEPSVGTSQQTQG 68

X7 1 -MRARKWVADSLAMG------NLLARWNEEPSVGTSQQTQ------G 34

X8 1 ------MG------NLLARWNEEPSVGTSQQTQ------G 22

X9 1 -M------EPST------G 05 Figure 3.3 Sequence alignment of the isoforms of chicken BCO2 using COBALT. Sequence alignment of the five isoforms of chicken (Gallus gallus) BCO2 listed in Pubmed (180) and the recombinant chicken BCO2 used in this study, generated by COBALT (http://www.ncbi.nlm.nih.gov/tools/cobalt/) (178). The isoforms differ only in the N-terminal sequences. The G in red background denotes the start of sequence identity. Tag- His-tagged recombinant chicken BCO2 in this study, X5-X9- isoforms of chicken BCO2 listed in Pubmed.

Figure 3.4 Cleavage of β-carotene by purified recombinant chicken BCO2. Chromatograms from the reaction mixture of β-carotene (20 μM) and BCO2 at 37°C using A) 15 min reaction time and 5 μg BCO2/200 μL, B) 30 min reaction time and 25 μg BCO2/200 μL and C) 120 min reaction time and 25 μg BCO2/200 μL. The product peak (*) has the same retention time and UV-Vis spectrum as standard β-apo-10′- carotenal, and was also verified by LC-MS. Chromatograms for standard β- apocarotenoids (β-apo-13-carotenone, retinal, β-apo-14′-carotenal, β-apo-12′-carotenal, β-apo-10′-carotenal, and β-apo-8′-carotenal) are shown for reference (D). Only β-apo-10′- carotenal was formed enzymatically. Chromatography was performed using Zorbax Eclipse XDB-C18 LC-Column (4.6 x 50 mm, 1.8 μm, Agilent) and HPLC Method A described in section 2.2.. Signal monitored: 453 nm.

Figure 3.4

Figure 3.5 Cleavage of α-carotene by purified recombinant chicken BCO2. Chromatograms from the reaction mixture of α-carotene (20 μM) and BCO2 at 37°C using A) 15 min reaction time and 5 μg BCO2/200 μL, and B) 30 min reaction time and 25 μg BCO2/200 μL. As with β-carotene, β-apo-10′-carotenal was detected as product (peak 2). Peak 1 is the alternative cleavage product α-apo-10′-carotenal. Chromatograms for standard β-apocarotenoids (β-apo-13-carotenone, retinal, β-apo-14′-carotenal, β-apo- 12′-carotenal, β-apo-10′-carotenal, and β-apo-8′-carotenal) are shown for reference (C). Chromatography was performed using Zorbax Eclipse XDB-C18 LC-Column (4.6 x 50 mm, 1.8 μm, Agilent) and HPLC Method A described in section 2.2. Signal monitored: 453 nm.

Figure 3.5

Figure 3.6 Cleavage of β-cryptoxanthin by purified recombinant chicken BCO2. Chromatograms from the reaction mixture of β-cryptoxanthin (20 μM) and BCO2 at 37°C using A) 15 min reaction time and 5 μg BCO2/200 μL, B) 30 min reaction time and 25 μg BCO2/200 μL and C) 120 min reaction time and 25 μg BCO2/200 μL. As with β- carotene, β-apo-10′-carotenal was detected as product (peak 3). The alternative cleavage product, 3-hydroxy-β-apo-10′-carotenal (peak 1) has been verified by LC-MS. LC-MS analysis verified the identity of 3-hydroxy-β-apo-8′-carotenal (peak 2). Chromatograms for standard β-apocarotenoids (β-apo-13-carotenone, retinal, β-apo-14′-carotenal, β-apo- 12′-carotenal, β-apo-10′-carotenal, and β-apo-8′-carotenal) are shown for reference (D). Chromatography was performed using Zorbax Eclipse XDB-C18 LC-Column (4.6 x 50 mm, 1.8 μm, Agilent) and HPLC Method A described in section 2.2. Signal monitored: 453 nm.

Figure 3.6

Figure 3.7 UV-Vis spectra of β-apo-8′-carotenal, β-apo-10′-carotenal, and their putative 3-hydroxylated counterparts produced from the reaction of chicken BCO2 with β-cryptoxanthin. The λmax is shown for each spectrum. The peak numbers correspond to the chromatogram peaks identified in Figure 3.6. Peak 3 has the same UV- Vis spectra and retention time as standard β-apo-10′-carotenal. Peak 1 elutes much earlier but has the same λmax as β-apo-10′-carotenal. This is consistent with its LC-MS identification as 3-hydroxy-β-apo-10′-carotenal by LC-MS. Peak 2 elutes after peak 1, but much earlier than peak 3, and the UV-Vis spectrum is similar to that of standard β- apo-8′-carotenal. This is consistent with its LC-MS identification as 3-hydroxy-β-apo-8′- carotenal by LC-MS.

Figure 3.8. Cleavage of lutein by purified recombinant chicken BCO2. Chromatograms from the reaction mixture of lutein (20 μM) and BCO2 at 37°C using A) 15 min reaction time and 5 μg BCO2/200 μL, B) 30 min reaction time and 25 μg BCO2/200 μL and C) 120 min reaction time and 25 μg BCO2/200 μL. The cleavage lutein should produce 3-hydroxy-α-apo-10′-carotenal and 3-hydroxy-β-apo-10′-carotenal, but the chromatography used for panels A-C (using HPLC Method A described in section 2.2.) was not able to separate the two compounds. However, they were separated by the alternative solvent system described in the Materials and Methods section of this chapter (D). Signal monitored: 453 nm.

Figure 3.9. Cleavage of zeaxanthin by purified recombinant chicken BCO2. Chromatograms from the reaction mixture of zeaxanthin (20 μM) and BCO2 at 37°C using A) 15 min reaction time and 5 μg BCO2/200 μL, and B) 30 min reaction time and 25 μg BCO2/200 μL. As with lutein and β-cryptoxanthin, BCO2 cleavage produces 3- hydroxy-β-apo-10′-carotenal Chromatography was performed using Zorbax Eclipse XDB-C18 LC-Column (4.6 x 50 mm, 1.8 μm, Agilent) and HPLC Method A described in section 2.2. Signal monitored: 453 nm.

Zeaxanthin

OH O O HO

3-hydroxy-β-apo-10′-carotenal 3-hydroxy-β-ionone

O O O HO

3-hydroxy-β-ionone rosafluene dialdehyde

Figure 3.10. Cleavage of carotenoids by murine BCO2 at the 9-10 and 9′-10′ bonds to yield rosafluene dialdehyde.

Chapter 4: The human enzyme that converts dietary provitamin A carotenoids to

vitamin A is a dioxygenase*

*dela Seña, C., Riedl, K. M., Narayanasamy, S., Curley, R. W. Jr. Schwartz, S. J., and Harrison, E. H. (2014). The human enzyme that converts dietary provitamin A carotenoids to vitamin A is a dioxygenase. J. Biol. Chem. Published online March 25, 2014. 83

ABSTRACT

β-Carotene 15-15′-oxygenase (BCO1) catalyzes the oxidative cleavage of dietary provitamin A carotenoids to retinal (vitamin A aldehyde). Aldehydes readily exchange their carbonyl oxygen with water, making oxygen labeling experiments challenging.

BCO1 has been thought to be a monooxygenase, incorporating oxygen from O 2 and H2O into its cleavage products. This was based on a study that used conditions that favored oxygen exchange with water. We incubated purified recombinant human BCO1 and β-

16 18 18 16 carotene in either O2-H2 O or O2-H2 O medium for 15 minutes at 37°C, and the relative amounts of 18O-retinal and 16O-retinal were measured by liquid chromatography- tandem mass spectrometry (LC-MS/MS). At least 79% of the retinal produced by the reaction has the same oxygen isotope as the O2 gas used. Together with the data from

18 16 16 18 O-retinal-H2 O and O-retinal-H2 O incubations to account for non-enzymatic oxygen exchange, our results show that BCO1 incorporates only oxygen from O2 into retinal. Thus, BCO1 is a dioxygenase.

4.1 Introduction

Vitamin A deficiency is the most common vitamin deficiency in the world and affects an estimated 190 million preschool-age children and 19.1 million pregnant women worldwide (181). In areas of endemic vitamin A deficiency, people obtain vitamin A almost exclusively as provitamin A carotenoids found in foods of plant origin

(3). Provitamin A carotenoids are enzymatically converted to retinal (vitamin A 84 aldehyde) (Figure 4.1A) by the enzyme β-carotene 15-15′-oxygenase (BCO1) (33).

Hence, understanding the mechanism and regulation of this enzyme is important.

The reaction mechanism, and consequently, the nomenclature of BCO1 and other carotenoid cleavage oxygenases (CCO’s) have been controversial (44,97,182). The first report of a CCO was made in 1965 by Goodman and Huang (41), who showed that β- carotene was converted to retinal by cell-free rat intestinal homogenates in the presence

3 of O2. The following year, the same group then showed using H labels that the hydrogens of the 15-15′ double bond of β-carotene (the site of oxidative cleavage) are retained during the enzymatic oxidation reaction, and proposed that the reaction most likely has a dioxygenase mechanism (183). However, the label “dioxygenase” should only be used when oxygen labeling experiments have clearly established that only oxygen from O2 is incorporated by the enzyme into its oxidative cleavage products.

BCO1 was given the Enzyme Commission (EC) number 1.13.11.21 in 1972, designating a dioxygenase (184), 29 years before the first report of an oxygen labeling experiment. A monooxygenase mechanism was proposed for BCO1 in 2001 (115). In that study, α- carotene, purified chicken BCO1 and horse liver alcohol dehydrogenase (HLADH) were

17 18 incubated in an 85% O2-95% H2 O environment. HLADH was used to form retinols from the aldehydes, which readily exchange their carbonyl oxygen with water (185). The resulting products (retinol and α-retinol) were purified by high-performance liquid chromatography and silylated. Using gas chromatography-mass spectrometry (GC-MS), the authors found virtually equal enrichment of 17O and 18O in both silylated retinols, suggesting a monooxygenase mechanism (Figure 4.1 B). However, it is possible that the

85 long reaction time (7.5 hours) and extensive processing favored oxygen exchange between the initial aldehyde products and the aqueous medium. Also, the HLADH reaction is reversible, and the enzyme displays dismutase activity (interconverting the aldehyde into alcohol and carboxylic acid) (97,186). This means that the aldehydes were never completely eliminated during the 7.5 hour incubation, and a significant amount of oxygen exchange with water may have occurred. Despite the inconclusiveness of this study, the enzyme’s EC number was changed to 1.14.99.36, classifying it as a monooxygenase (184), and subsequent literature has referred to the animal orthologs of the enzyme as β-carotene 15-15′-monooxygenase (BCMO1). Indeed, the National Center for Biotechnology Information named the gene BCMO1 (116).

To elucidate the reaction mechanism of human BCO1, we conducted multiple oxygen labeling experiments with minimal reaction and processing times to minimize oxygen exchange between retinal and water. Our results demonstrate that BCO1 is not a monooxygenase, but a dioxygenase.

4.2 Materials and Methods

18 18 Chemicals- β-Carotene (≥97%), all-trans-retinal (≥98%), H2 O (97% atom) and O2 gas

(99% atom) and Dowex 50WX4 were purchased from Sigma-Aldrich.

18 18 Synthesis of O-retinal- All-trans-retinal (20 nmol), H2 O (200 equivalents), 2 mL acetonitrile and 60 mg of Dowex 50WX4 (hydrogen form) were stirred at room temperature in a closed vial protected from light for 1.5 hours. This is based on the 86 method of Kawanishi et al. (187). The solids were removed by decantation, and the retinoids were then extracted with 3x2 mL hexanes. The hexane extracts were combined and stored at -80°C. The final product is 91% 18O-retinal as measured by LC-MS/MS

(analytical methods described in pp.89-91).

Freeze-drying of purified recombinant human BCO1- Purified recombinant human BCO1 was prepared according to our previously published method (169). The purified enzyme preparation catalyzed the oxidative cleavage of β-carotene with a Vmax = 197.2 nmol

−1 −1 retinal/mg BCO1 × h, Km = 17.2 μm and catalytic efficiency kcat/Km = 6098 M min .

Ten μg of purified recombinant human BCO1 and 40 μL of 5x reaction buffer (500 mM

Tricine-KOH, pH 8.0 at 37°C, 2.5 mM dithiothreitol, 20 mM sodium cholate, 75 mM nicotinamide) (169) were combined in a 10-mL amber headspace vial, and the vial was capped and flash-frozen in liquid nitrogen. The headspace vials were stored in dry ice for

30 minutes during transport to the freeze-dryer. The caps of the headspace vials were then fitted with individual syringe needles for venting, and the vials were placed in the jar of the manifold freeze dryer (Labconco). Freeze-drying was done for 16 hours at 0.14 mBar. The syringe needles were removed, and the headspace vials were stored at -80°C until use. Each vial of freeze-dried enzyme produces about 60 pmol of retinal from 4 nmol of β-carotene with the in vitro BCO1 activity assay system described in the following section.

16 18 In vitro BCO1 activity assay in O2-H2 O- The in vitro enzyme assay using purified recombinant human BCO1 was based on our previously published method (169). The freeze dried enzyme-reaction buffer mixture in the headspace vial (described in previous

18 section) was dissolved in H2 O to a final volume of 160 μL and placed in a 37°C shaking water bath. The reaction was initiated by adding 40 μL of β-carotene substrate solution

(containing 4 nmol β-carotene, 0.3 μL Tween-40 and 20 nmol α-tocopherol) prepared in

18 H2 O. The reaction was allowed to proceed in the water bath with gentle shaking and the

16 vial exposed to air (which contains oxygen as 99.8% O2 (188)) for 15 minutes. The quenching with 37% formaldehyde in the original method had to be omitted since the

16 latter contains H2 O. Instead, the reactions were quenched with 300 μL of acetonitrile, and the lipophilic compounds were extracted with 3x1 mL of hexanes under red lights.

The combined hexane extracts were dried under N2, re-dissolved in 100 μL 3:1 (v/v)

18 acetonitrile-H2 O, filtered through a 0.22 μm syringe-driven filter, and injected into the

HPLC. The whole process from the start of the reaction to the elution of the retinal peak in the HPLC takes about 50-60 minutes.

18 16 In vitro BCO1 activity assay in O2-H2 O- The enzyme–reaction buffer solution (10 μg of purified recombinant human BCO1, 40 μL of 5x reaction buffer and water to a total volume of 160 μL) was placed in a headspace vial and degassed by exposure to water aspirator vacuum for 2 minutes. The headspace vial was purged with nitrogen gas, then

18 connected to the O2 gas cylinder and placed in a 37°C water bath. Forty μL of β-

16 carotene substrate solution, prepared in degassed water (H2 O), was then injected into

88 the vial using a syringe. The reaction was allowed to proceed in the water bath with gentle shaking for 15 minutes. The reaction was quenched by injecting 300 μL of

18 acetonitrile into the vial before the O2 gas flow was turned off. The reaction mixture was then extracted and processed as in the previous section, except that the extract

16 residue was re-dissolved in acetonitrile-H2 O.

Control experiments- To account for the oxygen exchange between water and retinal, we

16 18 incubated 60 pmol all-trans-retinal ( O-retinal) in the reaction mixture prepared in H2 O

16 containing active BCO1, as described above for “In vitro BCO1 activity assay in O2-

18 18 H2 O.” We also performed an analogous incubation using our synthesized O-retinal in

16 H2 O.

UHPLC-MS/MS method- The reaction mixture was separated by an Agilent 1290

UHPLC system (Agilent Technologies) using a Zorbax Extend 2.1 x 50 mm, 1.8 μm C18 column (Agilent Technologies). The flow rate was 0.8 mL/min, and the column temperature was 40 °C. The composition of solvents was as follows: A = 0.1 % formic acid in water; B = 0.1% formic acid in acetonitrile. A linear eluting gradient was applied as follows: isocratic 60% B for 0.5 min, gradient from 60-78%B over 3 minutes, gradient from 78-100%B over 1.5 min, isocratic 100% B for 2 min and re-equilibration to 60%B over 2 minutes.

The HPLC was interfaced with an Agilent 6550 Q-TOF mass spectrometer

(Agilent Technologies) using an electrospray ionization (ESI) source operated in positive

89 ion mode. The MS instrumental parameters included: sheath gas temperature, 400°C; flow rate, 12 L/min; drying gas temperature, 150°C; flow rate, 15 L/min; 3 Hz MS/MS acquisition; 10 Hz MS reference scans; 30 psig nebulizer; Vcap, 2000 V; nozzle voltage,

2000 V; fragmentor, 350 V; ion funnel settings for small molecules. MS/MS transitions were acquired by collision-induced dissociation (CID) of all-trans-retinal standard

(m/z=285.218) and 18O-retinal (m/z=287.226) and found to optimize at a collision energy of 7.5 eV. Source and CID gas was high purity (>98%) nitrogen. Calibration was performed using ESI-L tuning mix (Agilent Technologies G1969-85000) and within-run reference compound was hexakis (1H, 1H, 3H-tetrafluoropropoxy) phosphazine, m/z

922.010 (Agilent Technologies HP-0921).

Quantification of retinal oxygen isotopologues- The fragmentation patterns of 18O-retinal and 16O-retinal are virtually the same (Figure 3). The parent retinals were not used for quantification to minimize errors arising from other naturally occurring isobaric species, which constitute about 2.2% based on the natural abundance of 13C (188). MS/MS was used to discriminate between the retinal analytes from these isobaric species, which will give different fragmentation patterns.

For quantification of the retinal oxygen isotopologues, the MS/MS fragments m/z= 119.086, 175.150, 105.070, 133.101, 163.101, 195.163, 231.162, for 18O-retinal and m/z= 119.086, 175.143, 105.070, 133.101, 161.092, 193.159, 229.158 for 16O-retinal were summed to generate one extracted ion chromatogram for each parent retinal species.

These daughter ions were selected because they were the dominant fragment ions. Also,

90 the last three daughter ions listed for 18O-retinal and 16O-retinal differ by 2 amu, indicating that these fragments bear the oxygen atom.

4.3 Results

16 18 For the BCO1-β-carotene reaction in O2-H2 O medium, the retinal product obtained (about 60 pmol) after a 15-minute reaction was only about 3-10% 18O-retinal

(Figure 4.1C). This range reflects what we obtained from three experiments done on different days. A sample LC-MS chromatogram is shown in Figure 4.2A. The small

18 16 18 relative amount of O-retinal we observed in O2-H2 O medium suggests that BCO1 is a dioxygenase (Figure 4.1B). If the enzyme is a dioxygenase, then theoretically, it should produce only 16O-retinal, and the 18O-retinal we observed was due to oxygen exchange

16 18 with water. To verify this, we incubated 60 pmol of O-retinal with BCO1 in H2 O under the same conditions. The % 18O-retinal formed was similar (5-13%) to that produced in the reaction of BCO1 and β-carotene (Figure 1C). This confirms that the 18O- retinal we were detecting was coming from the oxygen exchange of retinal with water, and not from the enzyme incorporating oxygen from water during the oxidative cleavage reaction.

18 16 We then conducted the BCO1-β-carotene reaction in O2-H2 O medium.

Consistent with our previous experiments, the majority of the retinal product obtained

18 contains the same oxygen isotope as that of O2 (79-85% O-retinal). As in the previous section, this range reflects what we obtained from experiments done on different days. A sample LC-MS chromatogram is shown in Figure 4.2B, and the MS/MS traces for m/z= 91

285.218 and 287.226 (corresponding to 16O-retinal and 18O-retinal, respectively) are shown in Figure 4.3. To verify that the 16O-retinal (15-21%) we observed was due to oxygen exchange with water, we also incubated 18O-retinal (91% 18O-retinal) with BCO1

16 18 in H2 O under the same conditions. We observed 67-84% O-retinal, corresponding to a

7-24% net exchange (Figure 4.1C). Consistent with the previous section, these values

18 16 18 strongly suggest that BCO1 reacts with β-carotene in an O2-H2 O to form only O- retinal, and the small relative amount of 16O-retinal is due to oxygen exchange with water.

16 18 We also performed the BCO1- β-carotene incubation in O2-H2 O for 7.5 hours, and the retinal product obtained was 50% 18O-retinal, which verifies that such a long incubation time will indeed lead to a false identification of the enzyme as a monooxygenase.

16 18 18 16 The O2-H2 O and O2-H2 O experiments strongly suggest that BCO1 incorporates only oxygens from O2 into retinal formed from the oxidative cleavage of β- carotene, and the minor amount of retinal with the same oxygen isotope as water is formed by non-enzymatic oxygen exchange. Thus, BCO1 is a dioxygenase and not a monooxygenase as had been previously thought.

If the parent retinals are used for quantification, the values differ by only 0-6% from the MS/MS calculation (Table 4.1), and the data still lead to the same conclusion that BCO1 incorporates only oxygen from O2 into retinal formed from oxidative cleavage of β-carotene.

4.4 Discussion

At this point, there is a very limited amount of literature on other CCO’s with which to compare our results. Most of the functionally characterized CCO’s are from plants, and these enzymes have been called “dioxygenases” despite the lack of conclusive oxygen labeling experiments (44,97). This error can be traced back to the lignostilbene

“dioxygenases.” As of 1997, these enzymes were called as such even though no oxygen labeling experiments were carried out (97,189-193). At best, these studies showed that these enzymes require O2. This error in naming was propagated into the CCO’s in 1997, when the first CCO to be cloned and characterized, maize Viviparous 14, was called a dioxygenase based on its sequence similarity to lignostilbene “dioxygenase” and not on oxygen labeling experiments (43,194). Even if the lignostilbene oxygenases were truly established to be dioxygenases back then, a sequence similarity is not necessarily a substitute for oxygen labeling experiments. Interestingly, the first report of an oxygen labeling experiment for a stilbene oxygenase (which was also identified because of sequence similarity to the plant CCO’s) showed a monooxygenase reaction mechanism

(195).

Of the more than 200 putative CCO’s to be found in sequence databases (44), there are only four other oxygen labeling experiments done apart from the aforementioned

2001 BCO1 study. The oxygen labeling experiments on water-stressed leaves of

Xanthium strumarium in 1984 (196) looked at only one cleavage product, and the

Arabidopsis thaliana study in 2006 (194) was deemed inconclusive because of the failure to show a consistent labeling pattern for the two cleavage products (Kloer and Schulz

93 give a detailed critique of these two studies (44)). An oxygen labeling experiment was done with Microcystis PCC 7806 cells, which generate β-cyclocitral and crocetindial

16 from oxidative cleavage of β-carotene (102). However, the results between the O2-

18 18 16 H2 O and O2-H2 O incubations were contradictory, and the authors acknowledge that the longer processing time for crocetindial may have favored oxygen exchange. Another oxygen labeling study done with a purified recombinant marine bacterial CCO that also cleaves β-carotene to retinal also shows a dioxygenase mechanism (65), consistent with our results.

Unlike other enzyme names such as “isomerase” or “lyase”, the names

“dioxygenase” and “monooxygenase” both indicate a specific reaction mechanism. Thus, the mechanism should be elucidated first before the name of an oxygenase is assigned.

For oxygenases that yield aldehydes, oxygen exchange with water should be minimized and accounted for. BCO1 was called a dioxygenase in 1972 without an oxygen labeling experiment, and a monooxygenase in 2001 despite an inconclusive study. Our results demonstrate that BCO1 is a dioxygenase.

4.5 Table

% 18O-retinal 16 18 18 16 O2-H2 O O2-H2 O BCO1 + β-carotene BCO1 + β-carotene 3, 6, 10 79, 85 2, 6, 12 80, 80

BCO1 + 16O-retinal (≥97%) BCO1 +18O-retinal (91%, 89%)* 5, 7, 13 67, 84 11, 6, 15 71, 82

Table 4.1. Comparison of retinal quantification by MS and MS/MS. The values using the parent ions obtained from LC-MS are in regular text, and those obtained by using the summation of daughter ions obtained from LC-MS/MS are in italics. Quantification by either method supports our conclusion that the retinal generated by BCO1 from β- carotene contains the same oxygen isotope as that of O2.

4.6 Figures

Figure 4.1. Human BCO1 is a dioxygenase. Human BCO1 is a dioxygenase. A). The putative reaction mechanisms of BCO1. A monooxygenase incorporates an oxygen atom from O2 in one retinal molecule, and an oxygen atom from water into the other (115). A dioxygenase incorporates only atoms from O2 into the cleavage products (183). B) Theoretical percentages of 18O-retinal that will be obtained for oxygen labeling experiments with BCO1 as a monooxygenase and as a dioxygenase. C) Summary of results of oxygen labeling experiments with purified recombinant BCO1. The numbers separated by commas are the % 18O enrichment of the retinal product from individual 18 experiments done on different days. Due to limited supply of O2, only two BCO1-β- 18 16 carotene reactions were done in O2-H2 O. Retinal obtained from the BCO1-β-carotene reaction contains predominantly the same oxygen isotope as O2. Control incubation of 18 16 16 18 active BCO1 with O-retinal in H2 O and O-retinal in H2 O account for the oxygen exchange that occurred in the corresponding BCO1-β-carotene reactions. Thus, BCO1 incorporates solely oxygen from O2 during the oxidative cleavage of β-carotene, and is therefore a dioxygenase. The isotopic purity of the 16O-retinal standard is based on natural abundance of 16O (188) and verified by LC-MS/MS. Isotopic purity of synthesized 18O-retinal measured by LC-MS/MS.

A. Monooxygenase Dioxygenase

B. Theoretical results % 18O-retinal 16 18 18 16 O2-H2 O O2-H2 O Monooxygenase Dioxygenase Monooxygenase Dioxygenase 50 0 50 100

C. Experimental results % 18O-retinal 16 18 18 16 O2-H2 O O2-H2 O BCO1 + β-carotene BCO1 + β-carotene 3, 6, 10 79, 85 BCO1 + 16O-retinal (≥97%) BCO1 +18O-retinal (91%)* 5, 7, 13 67, 84 18 16 16 * BCO1 + O-retinal incubated in O2-H2 O.

Figure 4.1

A x105

0 1 1.5 2 2.5 3 3.5 4 4.5 Counts vs. Acquisition Time (min)

B x104 2.8

2.4

2.0

1.6

1.2

0.8

0.4

0 1 1.5 2 2.5 3 3.5 4 4.5 Counts vs. Acquisition Time (min) Figure 4.2. LC-MS chromatograms for the reaction mixture of BCO1 and β- 16 18 18 16 carotene in O2-H2 O and O2-H2 O. Purified recombinant human BCO1 (10 µg/200 µL) was incubated with β-carotene (20 µM) for 15 minutes at 37°C, and the reaction mixture was analyzed by LC-MS/MS. The LC-MS chromatograms from the reaction 16 18 18 16 mixture in A) O2-H2 O and B) O2-H2 O are shown. The traces shown are the sum of the fragments from the MS/MS fragmentation of 18O-retinal (m/z=287.226) (blue trace), and 16O-retinal (m/z=285.218) (orange trace). MS/MS fragments used for quantification are listed in Materials and Methods.

Figure 4.3. MS/MS traces for the fragmentation of 16O-retinal and 18O-retinal 18 16 obtained from the reaction of BCO1 and β-carotene in O2-H2 O. The MS/MS trace for 16O-retinal (m/z=285.218) is shown in the upper panel, and that for 18O-retinal (m/z=287.226) in the lower panel.

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