A Dissertation

Entitled

Porcine Leukocyte 12-

By

Shu Xu

Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry

Dr. Max O. Funk, Jr., Committee Chair

Dr. Douglas W. Leaman, Committee Member

Dr. Timothy C. Mueser, Committee Member

Dr. Steven J. Sucheck, Committee Member

Dr. Patricia R. Komuniecki, Dean College of Graduate Studies

The University of Toledo

May 2012 Copyright © 2012, Shu Xu This document is copyrighted material. Under copyright law, no parts of this document may be reproduced without the expressed permission of the author. An Abstract of

Porcine Leukocyte 12-Lipoxygenase

by

Shu Xu Submitted to the Graduate Faculty as partial fulfillment of the requirements for the Doctor of Philosophy Degree in Chemistry

The University of Toledo May 2012

Lipoxygenases are a class of non-, non-sulfur in polyunsaturated metabolism involved in the modulation of basic physiologic processes. The work described within this dissertation covers biochemical characterization of porcine leukocyte 12-lipoxygenase, and presents the crystal structure of its catalytic domain.

For the first time, the iron content of the full length protein was obtained as high as

0.94 atom per molecule. The loss of this iron atom from the protein was evident, as the pH declined from 5 to 4 without losing the native protein folding, by electrospray ionization mass spectroscopy measurements. The full length protein failed to yield crystals in the screening conditions using numerous site-directed mutagenesis experiments and multiple crystallization techniques.

As part of this study, the catalytic domain of porcine leukocyte 12-lipoxygenase was successfully expressed in E. coli cells. The crystal structure of porcine 12-lipoxygenase

iii catalytic domain was determined to 1.89 Å as a complex with its specific inhibitor,

4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) (PDB id: 3RDE). This represents the first structural description of the 12-lipoxygenase catalytic domain. The complex revealed a new one-open-end U-shaped channel for the natural substrate, , which is remarkably different from the inhibitor (RS75091) binding pocket of rabbit 15-lipoxygenase.

Crystallization of the 12-lipoxygenase catalytic domain with arachidonic acid was also performed. Only fragments of electron density in the were present in the structure of the substrate- complex, illustrating that the substrate is not conformationally fixed in the crystal and may be converted into its products and derivatives. In addition, an unbound form of the catalytic domain was observed in the crystal structure of a recombinant reconstituted lipoxygenase catalytic domain.

Almost no structural differences were observed between the bound and unbound forms suggesting that lipoxygenase catalytic domain has little structural fluctuation or conformational alteration upon the binding of the inhibitor or substrate.

This research resulted in detailed biochemical insights for recombinant porcine leukocyte 12-lipoxygenase and its catalytic domain. The crystal structures of

12-lipoxygenase catalytic domain revealed key structural features of this enzyme, and also provided a basis for understanding enzymatic .

iv To my wife, Hongyan Ma, for your endless love.

To my parents and parents-in-law, for your support and understanding the importance of higher learning.

To my lovely son, Shaun Xu, for the happiness you bring into my life. Acknowledgements

First, I would like to address special thanks to my advisor, Dr. Max Funk, for all his support, advice, endless patience and continuous encouragement during these years. I would also like to thank Dr. Douglas Leaman, Dr. Timothy Mueser, and Dr. Steven

Sucheck for their service in my advisory committee. I acknowledge Dr. Lawrence

Marnett at Vanderbilt University for providing the cDNA of 12-lipoxygenase and the inhibitor used in this dissertation. Thanks go to Dr. Timothy Mueser, Dr. Alexander

Pavlovsky and Dr. Leif Hanson for their assistances with X-ray crystallographic techniques; to Dr. Pannee Burckel for the measurements of atomic absorption; to Dr.

Wendell Griffith and Dr. Dragan Isailovic for electrospay ionization measurements. I would like to thank the staff members at LS-CAT at the Advanced Photon Source of

Argonne National Laboratory for their help with X-ray diffraction data collection. I would like to express thanks to former and current members of Dr. Funk’s group: Dr.

Allan Sharp, Johanna Rapp, Ilka Decker, Maureen Gibbs, Marie Miniear, Kathryn Guinta, and Waqar Arif. Thanks to electronics specialists Thomas Kina and Youming Cao. I also extend appreciation to our neighbors at the University of Toledo, Dr. Viola’s, Dr.

Mueser’s, and Dr. Leaman’s groups and all others for letting me using their equipment, as well as providing a wonderful work environment. vi Table of Contents

Abstract…………………………………………………………………………………...iii

Acknowledgements………………………………………………………………………vi

Table of Contents………………………………………………………………………vii

List of Tables……………………………………………………………………………xi

List of Figures…………………………………………………………………...………xii

List of Abbreviations……………………………………………………………………xv

1 Lipoxygenase: Structure, Biochemistry, and Biological Functions…………………1

1.1 Introduction…………………………………………………………………1

1.2 Structures of …………………………………………………4

1.3 Lipoxygenase catalytic reactions……………………………………………7

1.3.1 activity of lipoxygenases…………………………………7

1.3.2 Other enzymatic activities of lipoxygenases…………………………11

1.4 Metabolites from lipoxygenase reactions………………………………….12

1.4.1 pathway…………………………………………………15

1.4.2 pathway………………………………………………………17

1.4.3 pathway…………………………….………………………18

1.4.4 Peroxide reduction pathway…………………………………………20

vii 1.5 Biological functions of lipoxygenase metabolites…………………………21

1.5.1 Roles of through 5-LOX pathway……………………….21

1.5.2 Roles of metabolites via 12/15-LOX pathway………………………22

1.5.2.1 Biological functions of …………………………………23

1.5.2.2 Biological function of …………………………………23

1.5.3 Biological functions of HETEs and derivatives………………………24

1.6 Perspectives………………...………………………………………………25

2 Experimental Methods………………..……………………………………………..27

2.1 Mutagenesis of plasmids of porcine leukocyte 12-lipoxygenase………….27

2.2 Construction of plasmids of 12-lipoxygenase catalytic domain…………...28

2.3 Expression of 12-lipoxygenase and its mutants……………………………29

2.4 Expression of 12-lipoxygenase catalytic domain…………………….29

2.5 Expression of recombinant manganese 12-lipoxygenase catalytic

domain……………………………………………………………………29

2.6 Purification of 12-lipoxygenase and 12-lipoxygenase catalytic domain…..30

2.7 Lipoxygenase enzymatic assay……………………………………………31

2.8 Protein concentration determination……………………………………….31

2.9 Sodium dodecyl sulfate polyacrylamide gel electrophoresis………………32

2.10 Atomic absorption measurements…………………………………………32

2.11 Electron ionization mass spectroscometry measurements…………………33

2.12 Ellman’s tritration…………………………………………………………33 viii 2.13 Dynamic light scattering measurements…………………………………34

2.14 Optimization of crystallization conditions…………………………………35

2.15 Crystallization of HLCDS-OPP and Mn-HLCDS-OPP……………………35

2.16 Crystallization of HLCDS-AA complex…………………………………...36

2.17 Diffraction data collection and structure determination…………………36

3 Biochemical Characterization of Porcine Leukocyte 12-Lipoxygenase………….…38

3.1 Introduction………………………………………………………………38

3.2 Results……………………………………………………………………39

3.2.1 Expression and purification of 12-lipoxygenase………………………39

3.2.2 Iron content of 12-lipoxygenase………………………………………41

3.2.3 ESI-MS measurements of 12-lipoxygenase…………………………...42

3.2.4 Crystallization experiments of 12-lipoxygenase………………………43

3.2.5 Protein engineering mutation to enhance crystallizability of PLL……45

3.2.6 Crystal screening results of PLL mutants……………………………48

3.3 Discussion…………………………………………………………………49

4 Crystal Structures of 12-Lipoxygenase Catalytic Domain………..……………52

4.1 Introduction………………………………………………………………52

4.2 Results……………………………………………………………………53

4.2.1 Expression of 12-lipoxygenae catalytic domain………………………53

4.2.2 Crystallization of 12-lipoxygenae catalytic domain…………………..55

4.2.3 Data collection and structure determination of HLCDS-OPP ix complex………………………………………………………………56

4.2.4 Specific inhibitor in the structure of 12-lipoxygenae catalytic

domain…………………………………………………………………60

4.2.5 Expression of recombinant manganese 12-lipoxygenase catalytic

domain…………………………………………………………………62

4.2.6 Crystallization of Mn-HLCDS-OPP and HLCDS–AA complexes……62

4.2.7 Data collection and structure determination of Mn-HLCDS-OPP and

HLCDS-AA complexes……………………………………………….65

4.2.8 An inhibitor unbound form of 12-lipoxygenase catalytic domain from

the crystal structure of Mn-HLCDS-OPP complex…………………65

4.2.9 The position of AA in HLCDS-AA complex………………………….67

4.3 Discussion…………………………………………………………………68

4.3.1 Crystallization conditions for 12-lipoxygenase catalytic domain……68

4.3.2 Variation of helix α2 in mammalian lipoxygenases…………………...69

4.3.3 The iron sites of mammalian lipoxygenases…………………………71

4.3.4 The inhibitor binding pockets of mammalian lipoxygenases…………72

4.3.5 Implication for inhibition and enzymatic catalysis……………………73

4.3.6 Substrate/intermediate in 12-lipoxygenase………………76

5 Conclusions………………………………………………………………………….79

References………………………………………………………………………………..83

x List of Tables

1.1 Iron environment of lipoxygenases………………………………………………….6

2.1 Plasmid constructions of 12-lipoxygenase and 12-lipoxygenase catalytic

domain……………………………………………………………………………28

2.2 The pipetting map for an example of A/B gradient………………………………..35

3.1 Half-life time of DTT in potassium phosphate buffer……………………………..40

3.2 Constructed mutants of porcine leukocyte 12-lipoxygenase………………………48

4.1 Data collection and refinement statistics of HLCDS-OPP complex………………58

4.2 Data collection and refinement statistics of Mn-HLCDS-OPP and HLCDS-AA

complexes………………………………………………………………………….64

xi List of Figures

1-1 Major metabolic pathways of arachidonic acid metabolism………………………2

1-2 Formation of hydroperoxides by the action of lipoxygenases………………………2

1-3 Cartoon structure of LOX-1………………………………………………4

1-4 The proposed mechanism of dioxygenase activity of lipoxygenases……………….7

1-5 The hydrogen abstraction and oxygenation position in lipoxygenase dioxygenase

reaction……………………………………………………………………………10

1-6 Products of HPETEs by different LOXs…………………………………………11

1-7 The proposed mechanism of synthase of 5-LOX………………….12

1-8 Metabolism of PUFAs in the lipoxygenase pathway of plants…………………….13

1-9 The lipoxygenase pathways in mammals…………………………………………14

1-10 The leukotriene pathway in lipoxygenase metabolism……………………………17

1-11 The lipoxin pathway in lipoxygenase metabolism…………………………………18

1-12 The hepoxilin pathway via 12-lipoxygenase………………………………………20

2-1 Reaction of Ellman’s titration……………………………………………………...34

3-1 Iron standard concentration curve of atomic absorption…………………………41

3-2 ESI-MS profile of PLL at different pHs…………………………………………...42

3-3 The crystals from full length 12-lipoxygenase…………………………………….44

xii 3-4 The diffraction pattern of the crystal hit of full length 12-lipoxygenase…………..44

3-5 The structure of 4-(2-oxapentadeca-4-yne)phenylpropanoic acid…………………45

3-6 Needle crystals of OPP in blank control experiment via vapor diffusion method…45

3-7 in 12-lipoxygenase model…...... 46

3-8 Clusters of residues from the surface entropy reduction (SER) for PLL…………..47

3-9 Crystal hit of PLLS-OPP complex…………………………………………………49

4-1 SDS-PAGE of HLCDS purification………………………………………………54

4-2 An example of the UV assay of lipoxygenase catalytic domain…………………54

4-3 Initial crystal hit of HLCDS-OPP from crystal screen 2 #22……………………...55

4-4 Crystals of HLCDS-OPP after optimization and addition of the cryoprotectant…..55

4-5 The diffraction patterns of HLCDS-OPP complexes using different size

collimators…………………………………………………………………………57

4-6 Surface representation for one asymmetric unit of HLCDS-OPP complex…...... 59

4-7 Secondary structural features for 12-lipoxygenase catalytic domain……………59

4-8 The location of the OPP binding site in HLCDS-OPP complex…………………...60

4-9 The specific interactions between OPP and HLCDS………………………………61

4-10 The kinetics profile of Mn-HLCDS………………………………………………62

4-11 Crystals of Mn-HLCDS-OPP complex……………………………………………63

4-12 Crystals of HLCDS-AA complex………………………………………………….63

4-13 Alignment between the unbound form of Mn-HLCDS complex and the inhibitor

bound form from HLCDS-OPP complex…………………………………………..66 xiii 4-14 Apo lipoxygenase catalytic domain in the Mn-HLCDS-OPP complex……………66

4-15 Alignment of the HLCDS-OPP and HLCDS-AA complexes……………………...67

4-16 Electron densities in the active site of HLCDS-AA complex……………………...68

4-17 A comparison of the positions of helix 2 in the structures of the mammalian

lipoxygenases………………………………………………………………………70

4-18 A comparison of the iron coordination environments for the mammalian

lipoxygenases………………………………………………………………………71

4-19 A comparison of the positions of OPP in the complex with 12-lipoxygenase and

RS75091 in the complex with 15-lipoxygenase…………………………………...73

4-20 Modeled arachidonic acid in the U shaped channel………………………………75

4-21 Stereodiagram of the dioxygen pathway in the 12-lipoxygenase catalytic

domain……………………………………………………………………………75

4-22 Implication for lipoxygenase catalysis……………………………………………76

xiv List of Abbreviations

AA ………………... arachidonic acid AOC …………….… allene oxide cyclase AOS ……………… allene oxide synthase APS ………………. Advanced Photon Source

Bis-Tris …………… bis(2-hydroxyethyl)-amino-tris(hydroxymethyl)-methane Bis-Tris propane ….. 1,3-bis(tris(hydroxymethyl)methylamino)propane

CAPS …………….. N-cyclohexyl-3-aminopropanesulfonic acid cDNA …………….. complementary DNA CHES …………….. N-cyclohexyl-2-aminoethanesulfonic acid COX ……………… CYP …………….…

DLS ………………. dynamic light scattering DMSO …………… dimethyl sulfoxide DNA ……………… deoxyribonucleic acid DSC ……………… differential scanning calorimetry DTNB ……………. 5, 5'-dithio-bis(2-nitrobenzoic acid) DTT ……………… dithiothreitol

EPR ……………… electron paramagnetic resonance ESI-MS ………….. electrospray ionization mass spectrometry E. coli ……………. Escherichia coli

FLAP …………….. five lipoxygenase activating protein FPLC …………….. fast protein liquid chromatography

GSH ……………… reduced glutathione

HEDH ……………. hydroxyeicosanoid dehydrogenase HETE …………….. hydroxyeicosatetraenoic acid HLCDS …………... His-tagged LCD with C210S C292S mutation

xv HODE ……………. hydroxyoctadecadienoic acid HPETE ………….… hydroperoxyeicosatetraenoic acid HPL ………………. hydroperoxide HPODE …………… hydroperoxyoctadecadienoic acid HSP ………………. heat shock protein HX ………………... hepoxilin

IMAC …………….. immobilized metal ion affinity chromatography

KIE ………………. kinetic isotope effect

LB ………………… Lysogeny Broth LCD ………………. 12-lipoxygenase catalytic domain LCDS ……………... 12-lipoxygenase catalytic domain with C210S/C292S mutations LOX ………………. lipoxygenase LT ………………… leukotriene LTA4H …………….. leukotriene A4 LX ………………… lipoxin

MES ………………. 2-(N-morpholino)ethanesulfonic acid MLR ……………… mixed lymphocyte reaction Mn-HLCDS ……… manganese recombinant HCLDS

OD ……………….. optical density OPDA ……………. 9S,13S-12-oxophytodienoic acid OPP ………………. 4-(2-oxapentadeca-4-yne)phenylpropanoic acid oxo-ETE ………….. oxo-eicosatetraenoic acid

PCR ………………. ploymerase chain reaction PDB ………………. protein data bank PEG ……………… polyethylene glycol PLL ………………. porcine leukocyte 12-lipoxygenase POX ……………… peroxygenase PUFA …………….. polyunsaturated fatty acid

RMSD …………….. root mean square deviation

SDS-PAGE ………. sodium dodecyl sulfate polyacrylamide gel electrophoresis SER ………………. surface entropy reduction

TB ………………... Terrific Broth

xvi TCEP …………….. tris(2-carboxyethyl)phosphine Tris ……………….. tris(hydroxymethy)aminomethane TX ………………… trioxilin

UV ………………... ultraviolet

VLX ………………. vegetative soybean lipoxygenase

xvii Chapter 1

Lipoxygenases: Structure, Biochemistry and Biological

Functions

1.1 Introduction

Polyunsaturated fatty acid (PUFA) metabolism is involved in the biosynthesis of over one hundred compounds, serving a variety of important physiological functions in human beings (1). In particular, arachidonic acid (AA) functions as the major precursor for a number of molecules termed that have essential roles in human diseases, including type-1 and type-2 diabetes (2), atherosclerosis (3), Parkinson's disease (4),

Alzheimer's disease (5), and variety of cancers (6-8). In response to assorted stimuli from the biofluids, such as cytokines, peptides, and growth factors, arachidonic acid is released from the cell membrane by the action of phospholipases for instance, phospholipase A1 (9).

Cyclooxygenase (COX), lipoxygenase (LOX) and cytochrome P450 (CYP), are the main three families of in arachidonic acid metabolism (Figure 1-1), converting arachidonic acid into a number of eicosanoids including numerous important biofunctional molecules, such as leukotrienes (LTs), hydroxyeicosatetraenoic acids

1 (HETEs) and hydroxyoctadecadienoic acids (HODEs) from the lipoxygenase pathway, (PGs) including G2 and H2 as well as (TXs) through the cylooxygenase pathway, and various epoxides and HETEs from the cytochrome P450 monoxygenase pathway (1).

O

OH

Arachidonic acid

Lipoxygenase Cycloxygenase Cytochrome P450

HPETE

TXA2 EETs HETEs Leukotrienes, HETEs, PGI2 Hepoxilins, Lipoxins... PGE2

Figure 1-1 Major metabolic pathways of arachidonic acid metabolism. Three families of enzymes participate in the arachidonic acid cascade: lipoxygenase, cyclooxygenase, and cytochrome P450.

Ha/Hb Hb Ha +O2 R1 R2 R1 R2 LOX

OOH

Figure 1-2 Formation of hydroperoxides by the action of lipoxygenases.

Among these three families of enzymes, lipoxygenases are a class of non-heme, non-sulfur iron dioxygenases that carry out the stereo-specific peroxidation of polyunsaturated fatty acids containing at least one 1,4-pentadiene (Figure 1-2) (10, 11). 2 Lipoxygenases have been found including (12), fungi (13, 14), algae (15,

16), plants (17-20), and animals (21-23). The different mammalian lipoxygenases, the

5S-, 12S-, 12R-, and 15S- lipoxygenases, are named for the positional specificity of oxygenation where they oxygenate their polyunsaturated fatty acid substrates, with the use of stereoisomer designation (S and R) as appropriate (24). However, this classification is not optimal. Arachidonic acid is not the preferred substrate for many non-mammalian LOXs and with other substrate fatty acids (e.g. ) the reaction specificity can be quite different. LOX-isoforms can exhibit distinct reaction specificities in different conditions. With the advances of modern bioinformatics and molecular biology, various lipoxygenases have been identified, cloned, and expressed.

However, due to our inability to predict the reaction specificity of oxygenation based on the primary structure of these enzymes, the nomenclature of these newly discovered lipoxygenases is not appropriate.

In humans, LOX enzymes include leukocyte 5-LOX, 12-LOX (with and leukocyte forms), and 15-LOX (with reticulocyte/leukocyte 15-LOX-1 and epidermis

15-LOX-2) (25). Arachidonic acid is the preferred substrate and is a component of the membrane until liberation by phospholipases (26). The 5-LOXs are involved in the biosynthesis of the leukotriene inflammatory products (27). The leukotriene pathway is initiated with synthesis of leukotriene A4 by the action of 5-LOX.

In human, the leukocyte 12-LOX and 15-LOX can form similar products from common substrates and their pathways are related (28). In other animals, mice only express leukocyte 12-LOX (29), while rabbits express both reticulocyte 15-LOX and leukocyte

12-LOX (30). These expression variations among species and the specificity difference

3 among the make it difficult to translate data obtained from different animal models of diseases to their human counterparts.

1.2 Structures of lipoxygenases

Lipoxygenases are a class of monomeric non-heme iron enzymes that have a molecular mass of ~75-80 kDa in animals and ~94-104 kDa in plants (10). Most LOXs consist of a small N-terminal β-barrel domain and a much larger C-terminal α-helical domain (Figure 1-3). Several complete crystal structures of plant and animal LOXs are available, including soybean lipoxygenases (LOX-1, LOX-3, VLX-B, and VLX-D)

(31-33), coral 8R-lipoxygenase (34, 35), rabbit reticulocyte 15-lipoxygenase (36, 37) and most recently human stable 5-lipoxygenase (38). The overall appearance of these LOXs is quite similar, as the two well-defined domains are assembled in an ellipsoid or cylindrical shape (39).

In some lower organisms, LOXs were discovered as fusion proteins, in which the lipoxygenase domain was expressed with another catalytic domain that plays a role in (40). In the coral Plexaura homomalla, the LOX protein that produces 8R-HPETE is fused with a heme-containing domain which converts the fatty acid peroxide to an allene oxide (41, 42). Another example is an allene oxide synthase-lipoxygenase (AOS-LOX) fusion protein from coral Gersemia fruticosa, which shares 84% sequence identity with the P. homomalla AOS-LOX (43, 44). AOS-LOX fusion proteins have also been discovered in the cyanobacteria Anabaena PCC 7120 (45) and Acaryochloris marina (46, 47). It is out of the ordinary that some of the cyanobacteria isoforms only bear a truncated catalytically active lipoxygenase domain while lacking the N-terminal β-barrel domain (16).

4 Figure 1-3 Cartoon structure of soybean LOX-1 (PDB: 1YGE). The N-terminal domain (residues 1-146) is colored in pink, while the C-terminal domain (residue 147-839) is in slate. The iron atom is located in the C-terminal domain, shown as an orange sphere.

All the N-terminal β-barrel domains in the reported lipoxygenase structures have been observed as anti-parallel β-strands. This domain belongs to the C2-domain super family, implicated in membrane binding (48). For soybean LOX-1, the β-barrel domain is made of the first 146 amino acid residues (31). In the case of animal LOXs, the

β-barrel domains are formed from around the first 110 residues (34). The N- and

C-terminal domains are covalently linked by a randomly-coiled loop. Even though the

N-terminal domains of the soybean LOXs are significantly larger than those of animals, their overall appearances are very similar. The interface between these two domains amounts to about 1600 Å2 for the rabbit 15-LOX, while the plant LOXs give larger

5 inter-domain contacts (2600 Å2 for the soybean LOX-1) (39). Biochemical characterizations on the ‘‘mini-LOX”, a truncated LOX lacking the N-terminal domain obtained by limited trypsin digestion of soybean LOX-1 (49), and the preliminary study of the N-terminal domain truncation of 15-LOX (50) indicate that the N-terminal β-barrel domain is not necessary for catalytic activity, but may play a role in the regulation of turnover.

The C-terminal domain contains the active site where a single metal atom resides.

This atom is an iron in all lipoxygenases except those from fungi, where a manganese lipoxygenase has been reported (14, 51). The C-terminal catalytic domain of most

LOXs consists of α-helices and harbors the catalytically active non-heme iron. The iron is octahedrally coordinated by four amino acid side chains, the C-terminal carboxylic group of the polypeptide chain and one hydroxide or water molecule (Table 1.1). In the reported animal LOXs, the catalytic domain consists of around 21-23 helices, interrupted by a small β-sheet sub-domain. Two long central helices cross the C-terminal domain, adapting π-helix conformations that provide the residue ligands for the active iron site

(39). The binding pocket for the substrate is located in the C-terminal domain nearby the iron site, which has been proved by the structure of the “purple” soybean LOX-3 (52), and as well as the rabbit 15-LOX-inhibitor complex (37).

Table 1.1

Iron environment of lipoxygenases.

Enzyme Iron environment residues Soybean LOX-1 His-499, His-504, His-690, Asn-694, Ile-839 Soybean LOX-3 His-518, His-523, His-709, Asn-713, Ile-857 Rabbit 15-LOX His-361, His-366, His-541, His-545, Ile-663 Human 5-LOX His-367,His-372, His-550, Asn-554, Ile-673

6 1.3 Lipoxygenase catalytic reactions

LOXs are versatile and multifunctional enzymes, catalyzing at least three different reactions: oxygenation of substrates (dioxygenase reaction), secondary conversion of hydroperoxy (hydroperoxidase reaction) (53), and formation of epoxy leukotrienes

(leukotriene synthase reaction) (54).

1.3.1 Dioxygenase activity of lipoxygenases

The dioxygenase reaction is most prevalent in all LOXs, which has been characterized or proposed as several key steps: activation of the enzyme, hydrogen abstraction, addition of oxygen, combination of peroxy radical with iron, and the dissociation of the peroxide (Figure 1-4).

H

HOO Fe(III)-OH LOOH

H H H LH

ROOH Fe(III) OO Fe(II)-OH2 Fe(III)-OH "Purple"

H-Tunneling

H H

OO Fe(II)-OH2 Fe(II)-OH O2 2 Figure 1-4 The proposed mechanism of the dioxygenase activity of lipoxygenases (52).

It has been proven that lipoxygenase has the resting state where iron is in the inactive ferrous state (Fe2+) and the active ferric state (Fe3+) for the catalysis (55, 56). The lipoxygenase reaction cycle begins by the activation of the ferrous iron to the oxidized

7 ferric iron by trace hydroperoxides or free radical species in the environment. In most isolated lipoxygenases, a lag phase was observed after the addition of exogenous arachidonic acid (in most cases of animal LOXs) or linoleic acid (in most cases of plant

LOXs). It has been proved that the addition of lipid hydroperoxides shorten this lag phase in vitro (57). The ferrous iron in the resting state is coordinated with side chains of four amino acid residues and the C-terminal carboxylate as well. One molecule of water occupies the free sixth position of the octahedaral configuration of iron, which has been determined by high resolution X-ray crystallographic studies of soybean LOX-1

(58). This ferrous ion was oxidized by the lipid hydroperoxide to be ferric while the bound water was converted into a hydroxide bound.

Once the enzyme is activated, the first step of the catalytic cycle is the hydrogen abstraction from the methylene group of PUFA by a hydrogen tunneling mechanism (59).

This hydrogen abstraction turns out to be also the rate-determining step of the overall reaction. In this step, hydrogen abstraction is stereo-selective from a bisallylic methylene moiety of the fatty acid into a free radical; the iron is reduced back to the ferrous oxidation state due to the transfer of the electron to the metal. The hydroxyl group bound to the ferric ion in the active form of LOX constitutes a perfect hydrogen-abstracting moiety. Large kinetic isotope effects (KIEs) were observed in the lipoxygenase hydrogen abstraction supporting the H-tunneling mechanism (59, 60). In this model, a very short distance between the hydrogen atom of the methylene group and the hydrogen acceptor, hydroxide, is required for hydrogen abstraction (61).

After the hydrogen abstraction, a molecule of dioxygen reacts with the radical forming the peroxy radical. The stereo- and regio- selectivity of the product is governed

8 by the location of molecular oxygen under steric restraints of the active site. A single residue controlling the stereochemistry of the reaction has been identified as a highly conserved Ala in S lipoxygenases and a Gly in R lipoxygenases (62). The basis for R and S stereocontrol also involves a switch in the position of oxygenation on the substrate

(Figure 1-5). Another model that can explain the regiospecificity of LOX enzymes is the so-called substrate orientation model (63). According to this hypothesis, the site of specific oxygen insertion is dependent on the substrate orientation within the active site of the LOX. For example, in the case of linoleate 13-LOX, the fatty acid penetrates the active site with the methyl-end ahead, while for linoleate 9-LOX, an inverse substrate orientation is proposed where the fatty acid enters the enzyme with the carboxy-terminus first (“head-to-tail orientation”). Positioning of the correct pentadiene of AA and controlling which carbon is oxygenated after hydrogen abstraction is unique to each LOX isoenzyme (64).

After the addition of dioxygen, the peroxy radical is reduced by accepting an electron from the catalytic iron center via a proton coupled electron transfer mechanism and thus reactivating iron from the ferrous state to the ferric state. The peroxy group will combine with the ferric iron to form a “purple complex”, which has been documented both in solution and crystal states (52, 65). The last step of the reaction is to release hydroperoxides from the purple complex, and regenerate the complex of ferric ion and the bound hydroxide as the starting point of the cycle.

9 Fe-OH H Fe-OH H COOH A Fe-OH H H O H 2 H 5R 8R 9S 11R 12S 15S

Fe-OH Fe-OH H H Fe-OH H H H O2 H 15R B 12R 11S 9R HOOC 8S 5S

Figure 1-5 The hydrogen abstraction and oxygenation position in lipoxygenase dioxygenase reaction. The central C7, C10, C13 of the three pentadienes in AA are highlighted in colors for detailed analysis, and the corresponding Fe-OH complexes are highlighted in the same color. (A) If the hydrogen of the methylene carbon lies proximal to Fe-OH complex and is removed, di-oxygen addition will happen as the antarafacial addition rule states. (B) If the pentadiene is flipped or the Fe-OH complex lies on the other side of AA, the stereochemistry of products are opposite to the ones in (A).

10 OOH COOH COOH

HOO 5-HPETE 5-LOX P-12LOX 12-HPETE COOH

AA L-12LOX 15-LOX

COOH COOH

8-LOX

HOO OOH 12-HPETE 15-HPETE

+ OOH +

COOH COOH COOH

OOH HOO 15-HPETE 8-HPETE 12-HPETE Figure 1-6 Products of HPETEs by different LOXs (66).

1.3.2 Other enzymatic activities of lipoxygenases

Compared to the dioxygenase activity, the hydroperoxidase reaction of LOXs is relatively slow, but this activity would increase by using a suitable electron donor and co-substrate, for instance, H2O2 instead of lipid hydroperoxides (67). This activity has been shown to be involved in xenobiotic oxidation and may play some role in melanin formation (68) in some physiological situations (such as Parkinson’s disease). Although documented research indicates that the hydroperoxidase activity of LOX has a wide substrate spectrum, the detailed mechanism of this activity is not clear.

Other than the dioxygenase and hydroperoxidase activities, 5-lipoxygenase also exhibits the activity of leukotriene synthase. The production of leukotriene A4 (LTA4)

11 from the 5-HPETE as an intermediate utilizes the same iron active site and a similar catalytic cycle (Figure 1-7). This time the hydrogen is abstracted from the C10 of

5-HPETE forming a hexatriene radical. When the radical position is at C6, next to the hydroperoxy group, the iron donates one electron, and the oxygen-oxygen bond is broken homolytically forming the triene epoxide LTA4 and a free hydroxyl anion (69). This free hydroxide removes one proton from water (coordinated with iron) and restores the iron complex back to the ferric state. 5-LOX is the only enzyme known to have both the dioxygenase and leukotriene synthase activities to form the LTA4.

H H LTA4 10

6 5 OOH Fe(III)-OH O Fe(III)-OH

O Fe(II)-OH OH O OH H Fe(II)-O H H

Figure 1-7 The proposed mechanism of leukotriene A4 synthase of 5-LOX.

In some cases of 12S-lipoxygenase, the hydroperoxy group of 12S-HPETE is homolytically cleaved into an alkoxy radical initiating the formation of epoxyhydroxyl compounds, hepoxilins (HXs) due to an intrinsic HXA3 synthase activity (involving the hydroperoxidase chemistry) of 12S-LOX (70). However, a detailed mechanism of the hepoxilin synthase activity has not been proposed.

1.4 Metabolites from lipoxygenase reactions

Plant lipoxygenases convert linoleic acid (18:2) and (18:3) into a

12 variety of bioactive mediators involved in plant defense, senescence, seed germination, plant growth and development (71). On the other hand, animal lipoxygenases utilize arachidonic acid (20:4) as the natural substrate to generate numerous bioactive regulators in modulation of basic physiologic processes such as , , blood-clotting, and hypersensitivity reactions (10).

PUFA (18:2 or 18:3)

-DOX

dinor isoprostanes C18--hydro(pero)xy PUFAs C17-PUFAs

9-LOX 13-LOX

9S- or 13S-hydroperoxides

POX AOS

DES AOS octadecanoids

epoxy hydroxy PUFAs jasmonates LOX reductase divinyl ether PUFAs epoxy hydroxy PUFAs HPL

hydroxy PUFAs keto PUFAs

leaf aldehydes leaf alcohols traumation

Figure 1-8 Metabolism of PUFAs in the lipoxygenase pathway of plants (72).

In plants, the majority of accumulated hydroxy fatty acids and hydroperoxy fatty acids originate from the action of LOXs. Lipid peroxide-derived substances,

9S-hydroperoxy and 13S-hydroperoxy derivatives of PUFAs, are processed in four major

13 metabolic routes (Figure 1-8). (a) The peroxygenase (POX) pathway (72) converts hydroperoxide fatty acids to epoxy- or dihydrodiol polyenoic fatty acids. (b) In the allene oxide synthase (AOS) pathway, unstable allene oxides are formed from either nonenzymatic hydrolysis leading to α- and γ-ketols or enzymatic hydrolysis of chiral

9S,13S-12-oxophytodienoic acid (OPDA) by an allene oxide cyclase (AOC). (c) In the hydroperoxide isomerase (HPL) pathway, the oxidative cleavage of the hydrocarbon backbone of fatty acid hydroperoxides forms short chain aldehydes (C6 or C9) and the corresponding C12 or C9 fatty acids. (d) The DES pathway forms divinyl ethers such as colneleic acid or colnelenic acid (72).

In mammals, the metabolic pathway of lipoxygenase results in the production of bioactive lipids, such as leukotrienes, hepoxilins, trioxilins, , and protectins (73).

The predominant products of the major polyunsaturated fatty acids in mammalian cell metabolism, arachidonic acid and linoleic acid, are 5-HPETE, 12-HPETE, 15-HPETE and 13-HPODE (74-76). These metabolites can be further processed in the following four pathways (Figure 1-9). (a) In the peroxide reduction pathway, hydroperoxy fatty acids can be converted into the corresponding hydroxides by the action of glutathione . (b) In the epoxy-leukotriene pathway, a concerted action of the and hydroperoxidase activty of 5-LOX leads to the formation epoxy-leukotrienes such as

14,15-epoxyleukotriene A4. (c) The third pathway is called the lipoxin (LX) pathway, where 15-HPETE can be further dioxygenated by 5-LOXs, and then converted into tetraene-containing eicosanoids, lipoxins. In some other cases, 12-LOXs can generate lipoxins from the leukotrienes through 5-LOX metabolism. Other lipoxins, 15-epimer lipoxins, are also formed by the action of acetylated COX-2 and leukocyte 5-LOX (77).

14 (d) The fourth is the hepoxilin synthase pathway, where the hydroperoxy group is homolytically cleaved to an alkoxy radical initiating the formation of hepoxilins by the intrinsic hepoxilin synthase activity of 12-LOXs or non-enzymatic reactions (70).

HETEs

GSH peroxidase Leukotrienes 5-LOXs LOXs AA HPETEs 12/15-LOXs

Lipoxins 12-LOXs

Hepoxilins

Figure 1-9 The lipoxygenase pathways in mammals.

In addition to these pathways, in vivo and in vitro studies showed that the bicyclic diendoperoxides (named as hemiketal eicosanoids) were formed by the action of COX-2 using 5-HPETE in the cross-over pathway between COX-2 and 5-LOX (78).

1.4.1 Leukotriene pathway

With the assistance of the protein FLAP (79), 5-LOX translocates to the nuclear membrane and catalyzes the oxygenation of arachidonic acid to form the product

5-HPETE, which is converted into leukotriene A4 by the action of a dehydrase or 5-LOX itself (80). LTA4 is a central unstable intermediate in leukotriene biosynthesis (Figure

1-10) that may be transformed through the action of the enzyme leukotriene A4 epoxide

15 hydrolase (LTA4H) into . Alternatively, in the presence of synthase, leukotriene C4 could be produced as the glutathione adduct at the C6 position of leukotriene A4 (81). is formed by the cleavage of the glutamic acid moiety of LTC4 by γ-glutamyltranspeptidase once the leukotriene C4 is exported from the cytosol to the extracellular microenvironment. Cleavage of the glycine moiety from

LTD4 by a variety of dipeptidases results in the formation of .

Leukotrienes C4, D4 and E4 are all known as the cysteinyl leukotrienes (82).

Biosynthesis of LTB4, C4, D4 and E4 occurs predominantly in leukocytes, in response to a variety of immunological stimuli. LTB4 is an important chemical mediator in the development and maintenance of inflammatory reactions and disease states, such as rheumatoid arthritis, psoriasis and gout (83). Also, LTB4 demonstrated the ability to inhibit cell apoptosis (84), and has been shown to be procarcinogenic in several studies

(54, 85, 86).

16 O OOH O

OH 5-LOX OH

AA 5-HPETE

LTA4 synthase/5-LOX Lipoxin OH O 15-LOX/12-LOX O O OH LTC4 synthase OH S Cys Gly

LTA γ-Glu 4 LTC4

LTA4 hydrolase extracellular γ-glutamyl transpeptidase

OH OH O OH O

OH OH S Cys Gly LTB4 LTD4 LTB4-ω-hydroxylase dipeptideases OH OH O OH O

OH OH S OH Cys ω-OH-LTB4

LTE4

Figure 1-10 The leukotriene pathway in lipoxygenase metabolism (87).

1.4.2 Lipoxin pathway

Lipoxins are trihydroxyeicosatetraenoic acids with four conjugated double bonds in the structure, which are involved in the resolution phase of inflammation (88). Lipoxins are produced in leukocytes by the 15-LOX or 12-LOX pathway (Figure 1-11) (89).

15-HPETE (or 15-HETE) can be further dioxygenated by 5-LOXs into

15S-epoxytetraene, a tetraene-containing . This 15S-epoxytetraene can also be produced from LTA4 by 12-LOX (73). The epoxy intermediate, 15S-epoxytetraene is converted into lipoxin A4 or B4 by the action of lipoxin A4 or B4 hydrolase, depending on

17 the cell type (90, 91). During this process, the stereochemistry of the carbon

15S-hydroxyl group is retained. The epimer lipoxins are generated through the acetylated COX-2 and leukocyte 5-LOX pathways (92). The stereochemistry of the carbon 15R-hydroxyl group is retained as the stereochemistry of 15R-HPETE from acetylated COX-2 metabolism (93).

O

OH

AA acetylated COX-2

15-LOX 5-LOX, LTA4 synthase

O O O O OH OH OH

O(O)H 15S-H(P)ETE LTA4 OOH 15R-HPETE 15-LOX/12-LOX 15-LOX 5-LOX

O

COOH O

COOH

OH 15S-epoxytetraene OH 15R-epoxytetraene

HO OH OH HO OH OH COOH COOH COOH COOH

OH HO OH OH HO OH LXA4 LXB4 15-epi-LXA4 15-epi-LXB4

Figure 1-11 The lipoxin pathway in lipoxygenase metabolism (94).

1.4.3 Hepoxilin pathway

Hepoxilins are monohydroxyepoxy eicosanoids derived mainly from the product

12S-HPETE of 12-LOXs. These hepoxilins are produced in a number of organs or cell types, but they were found especially abundant in the epidermis in humans (95, 96).

18 Unlike the leukotrienes and lipoxins, the structure of hepoxilins contains hydroxyl groups and epoxy groups crossing C11-C12 without any conjugated double bonds. In the hepoxilin pathway, the hydroperoxide group of 12S-HPETE is isomerized by a hepoxilin synthase or 12-LOXs (Figure 1-12). Two hepoxilins have been characterized, hepoxilin

A3 (HXA3) and hepoxilin B3 (HXB3). Only HXA3 is biologically active (97). The epoxide ring is labile and can be opened by an epoxide hydrolase to yield trihydroxy metabolites, termed ‘trioxilins’’, which may also have some biological activity (95). In addition, another family of hepoxilins,14,15-hepoxilins and their cysteinyl adducts, were generated by the action of 15-LOX and cytochrome P450 (98, 99).

19 O

OH

AA

12-LOXs

O

OH

HOO 12S-HPETE

Hepoxilin synthase/12-LOXs

O OH O HO OH OH O O

HXA3 HXB3 Epoxide hydrolase OH O OH O HO OH HO OH

HO HO 8S-TXA3 8R-TXA3 Figure 1-12 The hepoxilin pathway via 12-lipoxygenase (70).

1.4.4 Peroxide reduction pathway

The HPETEs from the catalytic reaction of LOXs can be converted to the corresponding HETEs by the action of glutathione peroxidases at the expense of reduced glutathione cosubstrate in and leukocytes (100). It has been shown that glutathione peroxidase has the capacity of reducing hydrogen peroxide, free radicals and fatty acid hydroperoxides as well. The oxidation of HETE by hydroxyeicosanoid dehydrogenase (HEDH) converts HETE into oxo-eicosatetraenoic acids (oxo-ETEs)

20 (101). The latter have some physiological effects and could be further oxidized either through primary or secondary metabolism.

1.5 Biological functions of lipoxygenase metabolites

The metabolites from lipoxygenase catalysis are involved in a number of significant biological processes (10). In the 5-LOX pathway, 5-LOX catalyzes the first step in the biosynthesis of leukotrienes, which are pivotal lipid mediators of inflammation and allergy and also have important roles in cardiovascular diseases, cancer and osteoporosis.

On the other hand, 12/15-lipoxygenases generated predominately either 12-HPETE or

15-HPETE depending on the specific structural elements, which have a variety of functions in human tissues (73). For example, 12S-HPETE and 15S-HPETE are involved in monocyte binding in the vasculature, by stimulating protein kinase C and various cellular adhesion molecules (102). However, the metabolites via this pathway can have pro- or anti-inflammatory effects depending upon the tissues and species (103,

104).

1.5.1 Roles of leukotrienes through 5-LOX pathway

Leukotrienes exert their biological activities by binding to specific receptors.

Leukotriene B4 binds to the leukotriene , a G-protein-coupled receptor that predominantly transduces chemotaxis and cellular activation (105). The cysteinyl leukotrienes bind to two distinct receptors that have been identified as CysLT1 and

CysLT2 (106). CysLT1 is a membrane protein found in airway smooth muscle cells.

Stimulation of this receptor promotes signal transduction and causes smooth muscle contraction (107). The CysLT2 receptor was previously known as the LTC4 receptor.

Some research indicated that leukotrienes may also play a role in cardiovascular and

21 neuropsychiatric illnesses (108, 109). Leukotrienes also have a powerful effect in bronchoconstriction (110) and also increase vascular permeability of small blood vessels

(111). Cysteinyl leukotriene receptors, CysLT1 and CysLT2 are located on the cell membrane of mast cells, , and endothelial cells (112). Pro-inflammatory activities, such as endothelial cell adherence and chemokine production by mast cells, are stimulated upon the interaction between cysteinyl leukotrienes and their receptors (113).

Cysteinyl leukotrienes can induce asthma and other inflammatory disorders, reducing the airflow into the alveoli (114). Excess cysteinyl leukotrienes in the body could trigger anaphylactic shock (115).

To date, two types of therapies have been developed based on this pathway for asthma treatment (54). The inhibition of 5-LOX (e.g. ) is used to slow down or stop the synthesis of leukotrienes in response to the immune reaction. Several antagonists such as and are used to prevent cysteinyl leukotrienes from exerting their bronchoconstrictive effects. These classes of therapies have indicated an improvement in asthma parameters in some individuals with asthma (116).

1.5.2 Roles of metabolites via 12/15-LOX pathway

Several 12- and 15-LOX isoforms have been identified in mammalian cells including

12-LOX in platelets, and 12/15-LOX in vascular and immune cells. When arachidonic acid is metabolized, all of the different LOX isoforms generate HPETEs as the primary product. The latter are rapidly reduced intracellularly to the corresponding alcohols.

Alternatively, the hydroperoxides can serve as precursors for the generation of other classes of secondary lipid mediators, such as lipoxins, hepoxilins, and trioxilins in the

22 pathways of 12/15-lipoxygenase (97).

1.5.2.1 Biological function of lipoxins

Physiological roles of lipoxins and the response of lipoxin receptor activation are both species- and cell type- dependent (89). Lipoxins exert their function by binding to the lipoxin receptor (ALX) (117). In the subnanomolar ranges, lipoxins inhibit chemotaxis of and eosinophils (118), transmigration across both endothelial and epithelial cells (119), neutrophil diapedesis (120), and neutrophil entry into inflamed tissues in several animal models (121). Lipoxins have also exhibited inhibition of the growth of different cells, e.g. eosinophils and natural killer cells; but at the same time, they stimulate peripheral blood monocyte chemotaxis and adherence (122).

While lipoxins increase monocyte chemotaxis and adherence, these cells do not deregulate or release reactive oxygen species in response to lipoxins, suggesting that their actions are specific for locomotion and may be related to the recruitment of monocytes to sites of injury or inflammation (123). Hence, lipoxins are implicated to play a role in resolution or repair. Lipoxins can also promote relaxation of smooth muscle, including vasodilatory properties and promoting vasorelaxation (124). LXA4 has the ability to reverse precontraction of the pulmonary artery caused by F2α or endothelin

1 (124). Lipoxins are also potent inhibitors of peptidoleukotriene-stimulated vasoconstriction in glomeruli and bronchoconstriction in human asthmatic airways (120).

1.5.2.2 Biological function of hepoxilins

Hepoxilins, the bioactive metabolites generated mainly through 12-LOX pathways, exhibit interesting biological actions (125). In the skin, hepoxilins are found to be pro-inflammatory, but in neutrophils they are anti-inflammatory (99). HXA3 modulates

23 cytoplasmic calcium concentrations, by either stimulating intracellular calcium release

(126) or increasing calcium transport across the membrane (99). HXA3 also stimulates glucose-induced insulin secretion dose-dependently in rat pancreatic islets, causes hyperpolarization of both the membrane potential and the postsynaptic potentials in aplysia and mammalian neurons (127), and regulates the cell volume in platelets and neurons by activating K+ channels (70, 128). In addition, the migration of neutrophils across the intestines, the expression of heat shock protein 72, the release of arachidonic acid, and the formation of diacylglycerol, are also mediated by HXA3 (70).

1.5.3 Biological function of HETEs and its derivatives

The primary product HPETEs of LOXs can be converted to the corresponding

HETEs by glutathione peroxidases (129). Oxidation of HETEs by hydroxyeicosanoid dehydrogenase generates oxo-eicosatetraenoic acids (oxo-ETEs). HETEs and oxo-ETEs demonstrate different activities depending on the cells and tissues (130, 131).

Despite re-entering leukotriene synthesis, 5-HETE from the 5-LOX pathway can exhibit its biological function or be further oxidized into 5-oxo-ETE. It has been shown that 5-HETE can activate neutrophils independently of receptors (132). However, the oxidation product of 5-HETE, 5-oxo-ETE is a more potent mediator in the cell.

5-Oxo-ETE is also a chemoattractant for monocytes, stimulating the proliferation of prostate tumor cells (133). It is believed that the action of 5-oxo-ETE is mediated by

OXE receptor, a Gi protein-coupled receptor, which is highly expressed in cells (134).

Unlike 5-HETE, 12-HETE is associated with pathogenesis of hypertension (135), and may mediate angiotensin II (136) and TGF-β induced mesengial cell abnormality in

24 diabetic nephropathy (137). 12/15-LOX knockout mice were resistant to the induction of diabetes by low-dose streptozotocin (138) , implying that 12-HETE generation was cytotoxic to pancreatic β-cells. This is further supported by the observations of cytokine-induced production of 12-HETE in both islets and β-cell lines (139).

12-HETE may also contribute to mitochondrial and oxidative stress where β-cells are highly sensitive (140). For example, it has been shown that 12S-HETE increases the invasiveness and metastatic potential in prostate tumors and is involved in the carcinogenesis of prostate tumors (141). 12S-HETE is markedly elevated in psoriatic lesions and contributes to high blood pressure in pregnancy-induced hypertension (142).

More recently, an orphan G protein coupled receptor GPR31 was identified as the receptor for 12S-HETE (143). However, the details about the receptor in the signal pathway are not that clear.

15-HETE from the pathway of 15-LOX has been shown in vitro to inhibit LTB4 formation (144), and compete with 12-HETE formation and specifically inhibit the neutrophil chemotactic effect of LTB4. The mechanism of the inhibition of LTB4 formation is proposed to be due to modulation of the 5-LOX. In vivo, 15-HETE inhibits

LTB4-induced erythema and edema (145), and reduces LTB4 in the synovial fluid of carrageenan-induced experimental arthritis (146). 15-HETE has shown the potential of inhibition of the mixed lymphocyte reaction (MLR) (147), and induction of the generation of murine cytotoxic suppressor T cells (148). Interferon production in murine lymphoma cells was reduced by the action of 15-HETE (100).

1.6 Perspectives

Polyunsaturated fatty acids (especially arachidonic acid) and their lipid metabolites,

25 play essential roles in human health and diseases (149). In human beings, 5-, 12- and

15-lipoxygenases demonstrate a wide range of actions on multiple levels, from different systems to diverse organs, from various tissues to assorted cells (24). These functions of metabolites from lipoxygenases suggest various strategies for the therapy of these related diseases. The full understanding of the lipoxygenase pathways will be essential to establish the detailed action of these diverse lipid mediators. The biochemical characterization of the lipoxygenase isozymes will prompt structure-based drug design, and a better understanding of enzymatic catalysis and regulation of lipoxygenase activity.

The development of isoform-specific lipoxygenase inhibitors will be necessary to fully create the therapeutic opportunities to treat these disorders by suppressing the activities of certain correlated lipoxygenases.

26 Chapter 2

Experimental Methods

2.1 Mutagenesis of plasmids of porcine leukocyte 12-lipoxygenase

The plasmid pET-20b(+) bearing the cDNA of porcine leukocyte 12-lipoxygenase was a gift from Dr. Lawrence J. Marnett (Vanderbilt University). The mutant plasmids were constructed with the application of the QuickChange site-directed mutagenesis kit

(Strategene) following the manufacturer’s protocol. In general, the primers were designed based on the sequence of the DNA and the target mutation using an online primer design tool

(http://www.genomics.agilent.com/CollectionSubpage.aspx?PageType=Tool&SubPageType=

ToolQCPD&PageID=15). The QuikChange site-directed mutagenesis method was performed using PfuTurbo DNA polymerase in a thermal temperature cycler. Following temperature cycling, the parental DNA template was selectively digested by Dpn I endonuclease (target sequence: 5´-Gm6ATC-3´). The nicked vector DNA incorporating the desired mutations was then transformed into XL1-Blue supercompetent cells. The plasmids of desired mutations were isolated by a QIAprep Spin Miniprep Kit (Qiagen) and confirmed by a DNA sequencing service (Eurofins MWG Operon). All customized primers were synthesized by Invitrogen. All the plasmids constructed in this dissertation are summarized in Table 2.1.

27 2.2 Construction of plasmids of 12-lipoxygenase catalytic domain

The polymerase chain reactions were performed with customized primers using 5

PRIME MasterMix. The cDNA of the lipoxygenase catalytic domain (residues 112 to 663, with and without C210S/C292S mutations) were amplified by polymerase chain reaction using the plasmid pET-20-PLL and pET-20-PLLS as the templates. The PCR products were then cleaved with NdeI and XhoI, and were cloned into empty plasmid of pET-20b(+) or pET-28a(+) via NdeI/XhoI restriction sites.

Table 2.1

Plasmid constructions of 12-lipoxygenase and 12-lipoxygenase catalytic domain.

Constructions Plasmid Residue range and mutation Commentary pET-20-PLL pET-20b(+) Full length (residues 1-663) No-His tag pET-20-PLLS pET-20b(+) Full length, C210S, C292S No-His tag pET-20-PLLK pET-20b(+) Full length, K45A, E46A No-His tag Full length, E611A, E612A, pET-20-PLLG pET-20b(+) No-His tag E613A Full length, E266A, K267A, pET-20-PLLD pET-20b(+) No-His tag E268A Full length, K45A, E46A, pET-20-PLLKS pET-20b(+) No-His tag C210S, C292S Full length, C210S, C292S, pET-20-PLLGS pET-20b(+) No-His tag E611A, E612A, E613A pET-20-LCD pET-20b(+) C-terminus (residues 112-663) No-His tag C-terminus (residues 112-663), pET-20-LCDS pET-20b(+) No-His tag C210S, C292S C-terminus (residues 112-663), N-terminal pET-28-HLCDS pET-28a(+) C210S, C292S His tag C-terminus (residues 112-663), N-terminal pET-20-HLCDS pET-20b(+) C210S, C292S His tag

2.3 Expression of 12-lipoxygenase and its mutants

For the expression of full length protein or its mutants, the pET-20-PLL plasmids (or

28 PLL mutant plasmids) were transformed into Rosetta 2 (DE3) cells. Freshly grown bacterial cultures (1 mL) from a single colony were first grown in 100 mL Lysogeny Broth

(LB) containing ampicillin (100 µg mL−1) and chloramphenicol (34 µg mL−1) for 4 hr, and then the entire culture (OD600 ~ 1.2) was combined with 1000 mL Terrific Broth medium containing 100 µg mL−1 ampicillin and 34 µg mL−1 chloramphenicol, and the cells were incubated 24 hr at 30 °C with shaking at 200 rpm. The cells were collected by centrifugation and re-suspended in phosphate buffered saline and collected again by centrifugation.

2.4 Expression of 12-lipoxygenase catalytic domain

For the expression of lipoxygenase catalytic domain with and without His-tags, the pET-20-LCD (or pET-20-LCDS, pET-20-HLCDS) plasmids were transformed into Rosetta 2

(DE3) cells. A similar procedure was applied for the expression except that the expression medium was Auto-induction Lysgeny Broth (Auto-LB) medium (150) (10 g tryptone, 5 g yeast extract, 3.3 g (NH4)2SO4, 6.8 g KH2PO4, 13.4 g Na2HPO4·7H2O, 0.5 g glucose, 2.0 g

α-lactose, and 0.15 g MgSO4 dissolved in 1000 mL medium) with selective antibiotics ampicillin (100 µg mL−1) and chloramphenicol (34 µg mL−1).

2.5 Expression of manganese 12-lipoxygenase catalytic domain

For the expression of lipoxygenase catalytic domain with manganese, Rosetta 2 (DE3) cells bearing pET-20-HLCDS were used in the experiment. The cells were first grown in

M9 medium (M9 minimal medium supplemented with 0.5% glucose, 2 mM MgSO4, 100 μM

-1 -1 CaCl2, 0.001% thiamine-HCl, 150 μM of MnCl2, and 100 μg·mL ampicillin and 34 μg·mL chloramphenicol). After four passages, the cells were inoculated into 1000 mL M9 medium for expression, and the expression was auto-induced by α-lactose at a finial concentration of

29 1.5% (w/v) (150).

2.6 Purification of 12-lipoxygenase and 12-lipoxygenase catalytic domain

The cells were opened by sonication in 200 mL lysis buffer containing, 10 mM Tris-Cl,

1 mM TCEP, 60 µg mL−1 chicken egg white type II-O trypsin inhibitor, 100 µg mL−1 catalase,

20 µg mL−1 DNase I, pH 7.4 and 0.5 mg mL−1 chicken egg white lysozyme, and the supernatant of the lysate was obtain by ultracentrifugation at 33,000 g.

For the full length protein and its mutants, the protein of interest was precipitated using

40% saturated ammonium sulfate from the supernatant of the cell lysate. The ammonium sulfate precipitate was obtained by centrifugation at 4 oC at 3600 g for 30 mins. The pellet was dissolved in 10 mM Tris-Cl, 1 mM TCEP, pH 7.4 buffer and dialyzed against 10 mM

Tris-Cl, 1mM TCEP buffer overnight and further purified on HiPrep Q (GE Life Science) anion exchange column using 0 to 30% gradient of 10 mM Tris-Cl, 1 M NaCl pH 7.0. The active fractions from HiPrep Q were pooled and then further polished using Source 15Q (GE

Life Science) anion exchange column using the same gradient. For the catalytic domain without his-tags, the supernatant of the lysate was loaded directly onto a HiPrep QFF (GE

Life Science) anion exchange column. The fraction of interest was eluted using 0 to 30% gradient of 10 mM Tris-Cl, 1 M NaCl pH 7.0 and further purified on a Source 15Q (GE Life

Science) anion exchange column using the same gradient.

For the lipoxygenase catalytic domain with His-tag construction, the supernatant of the lysate was first loaded to a Ni-NTA column (bed volume ~5 mL), washed with 10 mL of 100 mM Tris-Cl pH 7.5, 20 mM imidozale, and then eluted out with 20 mL of 100 mM Tris-Cl pH 7.5, 200 mM imidozale by gravity. The elution was dialyzed against 10 mM Tris-Cl,

30 1mM TCEP buffer overnight and further purified on a Source 15Q anion exchange column using 0 to 40% gradient of 10 mM Tris-Cl, 1 M NaCl, pH 7.0.

All chromatographic procedures were conducted on an AKTA FPLC (Amersham

Biosciences) operated at 4 °C. An enzymatic assay was used to find the active fractions of the protein of interest and the purity of the isolated protein was evaluated by SDS PAGE in

10% Bis-Tris mini gels (NuPAGE, Invitrogen) using MES SDS running buffer.

2.7 Lipoxygenase enzymatic assay

The maximum rate of the lipoxygenase-catalyzed reaction with arachidonic acid as substrate was determined spectrophotometrically at 234 nm in 3 mL of assay buffer (50 mM

Tris-Cl, 0.03% Tween-20, pH 7.4) at 25 °C as previously described (151). Arachidonic acid was dissolved in absolute ethanol and was delivered by airtight 50 μL syringe. All final assay solutions contained 1% ethanol (v/v). The maximum reaction rate of lipoxygenase was obtained form the maximum value of the first derivative of UV absorbance at 234 nm against time by a program called Stivell created by former group member P.A. Braul (56).

2.8 Protein concentration determination

The protein concentrations were determined by the UV absorption at 280 nm carried out in a 1 mL curette. The protein samples were diluted 20~50 times dilution to get the OD value (600 nm) in the optimal range (0.2~0.8). For the full length protein and its mutants, a value for the molar extinction coefficient (ε) 117,210 M-1·cm-1 was used based on computational calculation (152). A value of 84,430 M-1·cm-1 was applied for the lipoxygenase catalytic domain constructions.

31 2.9 Sodium dodecyl sulfate polyacrylamide gel electrophoresis

(SDS-PAGE)

Each protein sample was prepared similarly by mixing 3 μL B-PER Reagent (Pierce), 10

μL of protein (0.5~1 mg·mL-1), 5 μL of NuPAGE LDS Sample buffer (4x), and 2 μL of 1 M

DTT. The molecular weight ladder sample was prepared in a similar way. All samples were heated for approximately 10 min in a 70 oC water bath, and the samples were then spun for 60 s in a micro centrifuge to pellet any precipitated protein. The electrophoresis running buffer was 1X NuPAGE MES SDS buffer (Invitrogen). To each well was added 10 μL of protein sample or molecular weight marker. The gel was run at 120 V for approximately 45 min. The gel was removed from the cassette and placed in a pyrex dish where it was rinsed with distilled water. After the water was decanted off, the Coomassie Blue stain was added to the gel and heated in a microwave for 1 min at high power. Then the gel was allowed to sit for at least 60 min with gentle agitation. The gel was then washed with aliquots of deionized water until all the excess stain was removed from the gel, and the bands could be seen clearly. Finally, the gel was placed between 2 sheets of cellulose soaked in the preserving solution containing 22.5% enthanol, 7.2% acetic acid and 10% glycerol and dried for preservation.

2.10 Atomic absorption measurements

The metal content determinations were carried out using a Perkin-Elmer Model 5100PC atomic absorption spectrophotometer in flame ionization mode. The flame gases were acetylene and air. Measurements for iron and manganese were conducted at 248.3 nm and

279.5 nm, respectively, with a slit width of 0.2 nm and a lamp current of 5 mA. A standard

32 curve was generated using certified atomic absorption standard iron and manganese reference solutions (1000 ppm ± 1%, Fisher Scientific). The standard solutions of Fe or Mn were diluted with HCl acidified deionized water to 10, 7.5, 5, 2.5 μg·mL-1. About 0.8 mL of standards or samples was injected into the flame for measurements. A blank buffer was also subjected to atomic absorption to correct for the background of the protein buffer. For each sample or standard, three measurements were obtained.

2.11 Electrospray ionization mass spectroscometry measurements

Electrospray ionization mass spectrometry on intact 12-lipoxygenase was performed on a Synapt HDMS (Waters) quadrupole time of flight ion mobility mass spectrometer. Ions of

12-lipoxygenase were generated using a nanoflow electrospray source with home-made nanospray emitters. Working samples of 12-lipoxygenase at 3 μM were prepared by dilution of a 600 μM stock solution (exhaustively desalted by dialysis with ammonium acetate buffer, 10 mM, pH 7.0) with ammonium acetate buffer (10 mM) adjusted to the appropriate pH. After dilution samples were equilibrated at room temperature for 30 min prior to mass spectrometry. The instrument parameters were maintained at the following constant values for each experiment: capillary voltage, 2.5 kV; sample cone, 40 V; cone gas,

0 L/h; trap collision energy, 6 V. Mass spectra were calibrated externally in the range m/z

2,506-68,000 using a solution of cesium iodide, and were processed using Masslynx 4.1 software (Waters). All mass spectra were averages of 300 scans and were presented unsmoothed and without background subtraction.

2.12 Ellman’s titration

The protein sample was dialysized into 50 mM Tris-Cl pH 8.5 buffer to remove any trace of reducing reagent. Ellman’s titration was carried out in the buffer of 50 mM Tris-Cl,

33 pH 8.5 (Figure 2-1). The surface accessible cysteines were confirmed with Ellman's reagent,

5, 5'-dithiobis-2-nitrobenzoic acid (DTNB) under nondenaturing conditions. After 5 min or

1 hr, the 2-nitro-5-thiobenzoate then was quantified by measuring the absorbance at 412 nm using an extinction coefficient of 14,150 M−1·cm−1 and the number of surface accessible cysteines was determined (153).

S S

Protein-Cys-SH + HOOC COOH

NO2 NO2 DTNB SH

Protein-Cys-S S NO2 +

COOH COOH NO2

TNB

Figure 2-1 Reaction of Ellman’s titration.

2.13 Dynamic light scattering measurements

Dynamic Light Scattering (DLS) is a technique for characterizing the size distribution of macromolecules in solution. This measurement can tell the homogeneity of protein molecules in solution. The DLS experiment was performed because it is particularly suited to determine if a protein solution is likely to crystallize or not. The protein sample with a concentration between 0.5 and 1.0 mg·mL-1 was first passed through a 0.02 µm filter and then centrifuged to avoid any aggregates during the DLS measurements. The experiment was carried out using a DynaPro™ (Protein Solutions™) Titan instrument from Wyatt

Technology and the data were analyzed using the Dynamics software version 7.3. The

34 measurements were made at 4 °C and 20 °C to determine which temperature was the best for the crystallization.

2.14 Optimization of crystallization conditions

When a protein crystal was found in the sparse-matrix screen, it was identified as a “hit”.

Then, the conditions of the crystallization “hits” were reproduced and optimized in an attempt to get larger single crystals using expansion trays. The “expansion trays” were prepared by the A/B gradient method using the sitting-drop vapor diffusion technique (154).

The A/B gradient method consists in varying a single chemical parameter (buffer, salt, additive concentration) around the crystallization “hit” condition. This gradient works with only two home-made solutions, solution A (low concentration), solution B (high concentration) and a standardized pipetting map (Table 2.2).

Table 2.2

The pipetting map for an example of A/B gradient (one row per experiment).

Well number 1 2 3 4 5 6 Solution A 1.0 0.8 0.6 0.4 0.2 0.0 Solution B 0.0 0.2 0.4 0.6 0.8 1.0

2.15 Crystallization of HLCDS-OPP and Mn-HLCDS-OPP

Purified protein was concentrated to 6 mg·mL-1 using Millipore Amicon 50 kDa MCO filtration devices and then incubated with solid OPP (0.5 mg per 200 μL protein sample) for

16 hours. This solution was then further concentrated and filtered to 11~13 mg·mL-1.

Crystals were grown by hanging-drop vapor diffusion method at 291 K by mixing 2 μL protein-inhibitor sample and 2 μL reservoir solution containing 0.1 M MES pH 6.5, 5~10%

35 PEG-20,000, 20% glycerol. The crystals were harvested after 5 days of growth and flash frozen for shipment and data collection.

2.16 Crystallization of HLCDS-AA complex

HLCDS-AA complex was obtained by a macro-seeding technique. Purified protein was concentrated to 12 mg·mL-1 using Millipore Amicon 50 kDa MCO filtration devices.

Arachidonic acid was delivered into the protein solution at the concentration of 0.15 mM (the final concentration of ethanol in the sample was 1%). Seed solution was prepared by mixing 0.5 μL of solutions containing needle looking crystals from HLCDS-OPP complex with 100 μL mother liquid without crashing the crystals. This seed solution was added to

HLCDS protein solution (12 mg·mL-1) at a ratio of 1:100 (v/v). Crystals were grown by hanging-drop vapor diffusion method at 291 K containing the same reagents as the crystallization conditions for HLCDS-OPP complex (0.1 M MES pH 6.5, 5~10%

PEG-20,000, 20% glycerol). Crystals were harvested after 10 days of growth.

2.17 Diffraction data collection and structure determination

Diffraction data of HLCDS-OPP complex were collected at 100 K at LS-CAT beamline

23-ID-D at the Advanced Photon Source, Argonne National Laboratory, Argonne, Illinois using a 20 μm beam collimator. HLCDS-OPP Data were processed with iMosflm and reduced by SCALA (155). Crystals belonged to space group P21. The C-terminal part of the rabbit 15-lipoxygenase structure (PDB ID 2P0M, residues 112 to 663 of chain A) was used as the search model. Molecular replacement was performed with Phaser for the

HLCDS-OPP complex (155). Mutations and manual model building were performed in

COOT (156). Refinement was continued in REFMAC with rigid body refinement and restrained refinement (157). was built using SMILE and waters were adding from

36 COOT (156). Ligand occupancy refinement was carried out in Phenix (158).

Ramachandran statistics were performed by PROCHECK (159) and illustrations were prepared with Pymol (DeLano Scientific L.L.C.).

Diffraction data of Mn-HLCDS-OPP and HLCDS-AA were collected at 100 K at

LS-CAT beamline 23-ID-G at the Advanced Photon Source using a 20 μm beam collimator.

For the data sets of HLCDS-AA and Mn-HLCDS-OPP, molecular replacements were accomplished in Molrep in CCP4 using the coordinates of HLCD-OPP as the model (155).

The refinement was continued in REFMAC with rigid body refinement and restrained refinement (157). Ligand was built using SMILE and waters were adding from COOT

(156). Ramachandran statistics were performed by PROCHECK (159).

37 Chapter 3

Biochemical Characterization of Porcine Leukocyte

12-Lipoxygenase

3.1 Introduction

12-Lipoxygenase, also called arachidonate 12-lipoxygenase, catalyzes the conversion of arachidonic acid to 12-hydroperoxy-5,8,10,14-eicosatetraenoic acid (12-HPETE). There are three isoforms of 12-lipoxygenase named after the cells where they are found: leukocyte, platelet and epidermis type-enzymes (160). It is evident that the tissue distribution of three

12-lipoxygenases is different from species to species (161). Even if the leukocyte and the platelet 12-lipoxygenase catalyze the same reaction, they differ by their tissue distribution, their substrate specificity, their primary structure and size.

Because the reticulocyte and leukocyte 12- and 15-lipoxygenases have closely related sequences and catalytic behavior, producing similar ratios of 12- and 15-hydroperoxides under comparable conditions, these isoenzymes are sometimes referred to as

12/15-lipoxygenases (73). For example, the subject of this dissertation, porcine leukocyte

12-lipoxygenase, is more like human leukocyte 15-lipoxygenase (86% identical) and rabbit reticulocyte 15-lipoxygenase (79% identical) than human platelet 12-lipoxygenase (66%

38 identical) or human 5-lipoxygenase (41% identical).

The porcine leukocyte 12-lipoxygenase (PLL) was first isolated in the late 1980s by immuno-affinity chromatography, and the reactivity of this enzyme with hydroperoxyeicosatetraenoic acids was studied (162). Modern cloning techniques advanced protein expression from insect cells (163), to yeast (164) and finally bacterium E. coli (165).

Marnett’s group also carried out the synthesis of inhibitors targeting 12-lipoxygenase (166) and later detailed studies of the inhibition mechanism of the specific inhibitor

4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) for leukocyte 12-lipoxygenase (152).

Several biophysical analyses were performed to characterize the protein in solution such as dynamic light scattering (DLS), differential scanning calorimetry (DSC), electron paramagnetic resonance (EPR) spectroscopy and mass spectrometry in our group by former colleague Johanna Rapp (151). This chapter presents some further biochemical characterization of porcine leukocyte 12-lipoxygenase. Some crystallization experiments were also conducted in order to obtain diffraction-quality crystals of porcine leukocyte

12-lipoxygenase.

3.2 Results

3.2.1 Expression and purification of 12-lipoxygenase

The pET-20b(+) plasmid bearing the cDNA for porcine leukocyte 12-lipoxygenase was transformed into Rosetta 2 (DE3) cells. Porcine leukocyte 12-lipoxygenase was expressed by incubating the cells at 30 oC for 24 hr using Terrific Broth taking the advantage of the leaky expression of pET plasmids. The sonication method was used to open the cells as described by Johanna Rapp (151). Once the cells were lysed, the lysate was ultra-centrifuged to remove the debris of the cells and collect the protein in the supernatant.

39 Then 40% saturation ammonium sulfate precipitation was used to further purify the protein of interest. The final pellet of ammonium sulfate could be further purified by chromatography or frozen at -20 oC for later purification. After dialysis of the ammonium sulfate pellet against 10 mM Tris-Cl, 1 mM TCEP buffer four times, the desalted solution was injected into an anion exchange column, HiPrep QFF, and further purified by another anion exchange column, Source 15Q.

Several changes were made to purify the porcine leukocyte 12-lipoxygenase based on

Rapp’s method. The reagent Tris-Cl was used to replace Bis-Tris propane (BTP) to maintain pH 7.0. In the former working buffer which contained 1mM dithiothreitol (DTT),

PLL only retained its activity for 3~5 days at 4 oC. DTT was not ideal for the stock solution of PLL as its recorded half-life time was relatively short from less than 1 hr to dozens of hr

(Table 3.1). Tris(2-carboxyethyl)phosphine (TCEP) was chosen as its thiol-free, odorless but a potent reducing reagent for the buffer.

Table 3.1

Half-life time of DTT in potassium phosphate buffer (167).

Conditions (all in 0.1 M potassium phosphate buffer) Half-life (hr) pH 6.5, 20 °C 40 pH 7.5, 20 °C 10 pH 8.5, 20 °C 1.4 pH 8.5 , 0 °C 11 pH 8.5, 40 °C 0.2 pH 8.5, 20 °C,+0.1 mM Cu2+ 0.6 pH 8.5, 20 °C, +0.1 mM EDTA 4

The specific activity of PLL did not change along with the modifications. The final calculated activity of the purified leukocyte enzyme was 8.14  0.59 μmol·mg-1·min-1 using 40 -1 -1 an A280 extinction coefficient of 117,210 cm M for the calculation of protein concentration

(152).

3.2.2 Iron content of 12-lipoxygenase

Iron plays a central role in the chemistry of the non-heme iron site in lipoxygenase catalysis. For biophysical analysis especially crystallization experiments, a homogenous sample would be critical and the iron content is a factor to judge the homogeneity of the sample as the previously determined values for this lipoxygenase were reported as “about

0.45” (162) and 0.70 ± 0.09 (168), or the value was not reported (152). The iron content determinations were carried out in the atomic absorption spectrophotometer in flame ionization mode using acetylene and air as flame gases. The iron concentration was calculated based on the standard curve of absorbance vs. iron concentration (Figure 3-1) measured on the same day and on the same device. In the experiment samples, a nearly stoichiometric amount of iron, 0.94  0.03 atoms per molecule, was found associated with the protein from the standard curve using authentic atomic absorption standard iron reference solutions (1,000 ppm ± 1%). Standard curve for Fe via AA

0.25

0.20

0.15

0.10

Absorbance y = 0.0225x + 0.0085 2 0.05 R = 0.9986

0.00 0.0 2.0 4.0 6.0 8.0 10.0 Fe standard concentration (µg/mL)

Figure 3-1 Iron standard concentration curve of atomic absorption (n=3).

41 Figure 3-2 ESI-MS profile of PLL at different pHs. (A) range 1000–6000 m/ z units, pH 3-10. (B) scale expansion around mass at m/ z 4168, pH 4–6.

3.2.3 ESI-MS measurements of 12-lipoxygenase

When electrospray ionization mass spectrometry was conducted on solutions of

12-lipoxygenase adjusted to different pH values, the mass spectra shown in Figure 3-2 were obtained. At pH 5.0 and below, the native ions were joined in the spectra by ions of lower mass, which is common for proteins undergoing acid-induced unfolding. Examining the individual ions more closely revealed that the native peaks lost mass corresponding to the mass of an iron atom (calculated mass difference, 58 amu is comparable to the actual mass of iron of 55.8 amu) as the pH was lowered from 6.0 to 4.0. These observations indicated that

12-lipoxygenase experienced a loss of iron upon the acid-induced unfolding with minimal structural alternation compared to the native form. This was quite different from similar experiments conducted on soybean lipoxygenases. 12-Lipoxygenase goes through acid-induced unfolding accompanied by the appearance of an iron-free intermediate that

42 retains a native conformation.

3.2.4 Crystallization experiments of 12-lipoxygenase

Dynamic light scattering and solubility screening of PLL demonstrated that in most buffers PLL behaved as a monomer with a relatively high solubility in low salt buffer.

Crystal screenings were conducted at both room temperature and 4 oC. Vapor diffusion and microbatch methods were applied for the crystallization in both robotic and manual set-ups using several commercial sparse matrix crystallization kits (including Crystal Screen, Crystal

Screen 2, Index (Hampton Research), Wizard I, Wizard II, Cryo I and Cryo II (Emerald

BioSystems), Anions, Cations, PACT (Qiagen)) and some home-made conditions.

Preliminary crystallization was not that successful. One of the reasons is that the native PLL has a relatively short half-life at both room temperature and 4 oC. Although the use of reducing reagent, such as TCEP, extended the half-life time of the protein in aqueous conditions, PLL failed to crystallize in any of the many different conditions from the sparse matrix screening kits. The screening results indicated that 12-lipoxygenase would be denatured if the pH of the buffer was lower than 5 as amorphous precipitations were observed; the protein precipitates were also found in the crystallization conditions with divalent ions even as low as millimolar level; skins were also observed in some cases by both vapor method and microbatch method indicating that the protein was denatured by those conditions.

Among all these conditions, we only found one crystal hit by hanging drop method in the condition of 0.1 M CHES pH 9.0, 10 % PEG-8,000 (Figure 3-3) at room temperature.

The protein in this condition was applied as 12 mg·mL-1 of protein in the buffer of 10 mM

Tris-Cl, 1 mM DTT, pH 7.4. Those crystals took almost two years to grow and were

43 diamond-shaped, with the longest dimension about 0.08 mm. However, these crystals did not diffract well in X-ray diffraction experiments (Figure 3-4). However, no crystals were found in the experiments of extensive optimization based on this condition.

Figure 3-3 The crystals from full length 12-lipoxygenase. Crystallization condition: 0.1 M CHES pH 9.0, 10 % PEG-8,000 at room temperature, PLL 12 mg·mL-1 in 10 mM Tris-Cl, 1 mM DTT, pH 7.4.

Figure 3-4 The diffraction pattern of the crystal hit of full length 12-lipoxygenase.

Crystallization was also examined by co-crystallization experiments with the

12-lipoxygenase inhibitor, 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP) (Figure 3-5) via multiple crystallization set-ups. DMSO and ethanol were used to solubilize this

44 hydrophobic chemical in aqueous buffer. However, due to the hydrophobicity of OPP, OPP tends to produce tiny fine needles in the crystallization experiments. This has been confirmed by blank control experiments where 2% DMSO was used to dissolve OPP (Figure

3-6).

O

COOH

Figure 3-5 The structure of 4-(2-oxapentadeca-4-yne)phenylpropanoic acid (OPP).

Figure 3-6 Needle crystals of OPP in blank control experiment via vapor diffusion method.

3.2.5 Protein engineering mutation to enhance crystallizability of PLL

For crystallization experiments, the stability of the protein is always one general consideration as the crystal trays usually sit at room temperature or 4 oC for a relatively long time period. To enhance the stability of proteins, one method is adding a reducing reagent 45 to the buffer. However, using DTT or TECP, PLL failed to crystallize under various conditions. Another strategy to extend the half-life time of the protein is the mutation of surface exposed cysteines to serines, as the oxidation of cysteines will cause the aggregation of protein. According to the model of PLL (generated by Dr. Allan Sharp based on the homology model of rabbit reticulocyte15-lipoxygenase), two cysteines (positions 210 and

292) were found on the surface of this model, and two cysteines (positions 38 and 600) were partially exposed to the accessible surface (Figure 3-7). Ellman’s titration under nondenaturing conditions suggested 3 to 4 free thiol groups (depending on the reaction time) present in PLL. Site directed mutagenesis was conducted to mutate cysteines 210 and 292 to serines. Mutant PLLS (C210S/C292S) showed less exposed free thiols (1 to 2) under the same nondenaturing condition in Ellman’s titration, while retaining a similar specific activity to the native enzyme.

Figure 3-7 Cysteines in 12-lipoxygenase model. The model of PLL based on rabbit 15-lipoxygenase is represented as surface. N-terminal domain is colored in pink, while C-terminal domain in cyan. All 14 cysteines (residue number 38, 78, 97, 210, 292, 346, 379, 443, 500, 527, 533, 560, 600) in the sequence were represented in yellow spheres, only residue 291 and 292 are fully exposed to the solvent while residues Cys38 and Cys600 are partially exposed.

46 Figure 3-8 Clusters of residues from the surface entropy reduction (SER) for PLL. The model of PLL based on rabbit 15-lipoxygenase is represented as cartoon. N-terminal domain of PLL is colored in pink, while C-terminal domain is colored in cyan. The predicted amino acid residues are in sticks. The iron is represented by a purple sphere. The clusters K, D, and G are color in orange, blue and red.

The other consideration is a surface entropy-reduction mutagenesis strategy which involves mutation of side chains on the surface of proteins, in order to reduce the entropic cost of forming ordered intermolecular crystal contacts and thus enhance the crystallizability of proteins (169). Typically, large and charged side chains, such as lysine and glutamate, are mutated to small and uncharged side chains, mostly alanines. Three clusters for mutagenesis were suggested by the SER (Surface Entropy Reduction Prediction Server) to predict sites that are most suitable for mutation designed to enhance crystallizability. Also, mutants including the combination of free cysteines to serines and two of these clusters were constructed (Figure 3-8). All mutants created so far are summarized in Table 3.2.

47 Table 3.2

Constructed mutants of porcine leukocyte 12-lipoxygenase.

Mutants Changed residues PLLS C210S, C292S PLLK K45A, E46A PLLG E611A, E612A, E613A PLLD E266A, K267A, E268A PLLKS K45A, E46A, C210S, C292S PLLGS C210S, C292S, E611A, E612A, E613A

3.2.6 Crystal screening results of PLL mutants

Crystal screenings of these mutants were also conducted at both room temperature and 4 oC by vapor diffusion and micro-batch methods against several commercial crystallization kits and home-made conditions. Small square-looking plate crystals were found in the crystal screening by hanging drop method. The protein in these crystals was the complex of protein PLLS mutant (concentration ~22 mg·mL-1) in the buffer of 10 mM Tris-Cl, 1mM

TCEP, 1 mM EDTA, 0.5% (w/v) octyl-β-D-glucoside, pH 8.0, and with the presence of 0.1 mM OPP (Figure 3-9). However, the diffraction pattern of these crystals using the in-house

X-ray source indicated that those crystals were likely to be small molecules instead of proteins, and citric acid tends to form crystals at 4 oC.

48 Figure 3-9 Crystal hit of PLLS-OPP complex. Crystallization condition: 0.1 M Citrate pH 5.5, 15% ethanol via hanging drop method at 4 oC, PLLS protein concentration is 15 mg·mL-1 OPP concentration is 1 mM (in 1 % DMSO).

3.3 Discussion

The full length protein was successfully expressed using a similar protocol according to a previous report (151). Approximately 40 mg of protein was successfully purified in a 3 liter scale expression. The iron content of full length protein was determined as 94% which indicates that the recombinant protein is fully intact and comparable to the protein isolated from porcine leukocytes. In the low pH conditions, the PLL is very likely to lose its iron but retain folding similar to the native protein. This mild acidic condition of pH 5 will be very useful for later metal reconstitution experiments. However, the stability of this metal free intermediate is still unknown. So far, all we know is that the ions were present in the solutions after 30 min at room temperature.

The DLS experiments demonstrated that the porcine leukocyte 12-lipoxygenase would

49 likely crystallize both at room temperature and at 4 °C. Crystal screens were tried at both temperatures to increase the chances to get protein crystals. In an attempt to avoid the heavy precipitate, the concentration of the protein was reduced in order to bring the precipitant/protein concentration to just below supersaturation (concentration of protein used as 6, 8, 10, 15, 20 mg·mL-1 in experiments), but the precipitate was still present in most crystallization conditions. On the other hand, clear drops were also observed in some of the basic conditions, such as CHES pH 9.5 and CAPS pH 10.0. Using these basic buffers as the working buffer for the protein, no crystals were obtained. Various crystallization techniques, such as co-crystallization, vapor diffusion, oil drop diffusion and microseeding, were applied with this condition but they resulted in the same observations. One crystal hit was obtained in the condition after a long time period. However, in repeated experiments, this condition failed to give any crystals. Mutants of PLL did not give crystals in various screening conditions. Mutations were mostly localized in the C-terminal domain. The working hypothesis for these mutations was based on eliminating surface exposed cysteines and reducing surface entropy.

In the future, the growth rate should be slowed to produce well-order crystals. Some new reagents to protect the unfolding process of the protein should be applied as only one crystal hit was obtained through a long time process. Crystallization is very tedious work that depends on many parameters so there are obviously still many experiments that could be tried to obtain good diffraction quality crystals. As lipoxygenases are also considered as membrane associated proteins, the crystallization screening kits should include the membrane protein kits. The choice of detergents in the purification and crystallization might be also applied for future crystallization experiments. Newcomer’s group mutated a lot of residues

50 on the loop of the N-terminal domains of coral 8R-lipoxygenase and human 5-lipoxygenase as the calcium ions, which are interacting with the N-terminal loops, change the conformation of the protein (38, 170). Their engineered proteins successfully produced diffraction quality crystals in both coral 8R-LOX and human 5-LOX (35, 38). Based on these observations, the alternative strategy for leukocyte 12-lipoxygenase constructions should be targeted on these N-terminal loops. The consideration of these calcium loops for the next generation of mutation will potentially benefit the crystallization experiments of

PLL. In addition, the flexibility between the N-terminal domain and C-terminal domain should also be taken into account.

51 Chapter 4

Crystal Structures of 12-Lipoxygenase Catalytic Domain

4.1 Introduction

The structure determination of mammalian lipoxygenases has been a struggle for many years. A complex between rabbit reticulocyte 15-lipoxygenase and its inhibitor was first determined in 1997 with some ambiguous assignments (36). Structural details of the interaction between the inhibitor and protein were unclear until a re-analysis of the original

X-ray diffraction data was published in 2008 (37). The revised crystal structure of

15S-LOX presents two proteins in the asymmetric unit, one with inhibitor and the other without, existing in two distinct forms (37). Very recently, with the deletion of putative membrane-insertion amino acids in the N-terminal domain and the modification of the

C-terminal anchor loop, the structure of a stable human 5-lipoxygenase mutant was successfully determined by X-ray crystallography (38). Besides these mammalian lipoxygenases, crystal structures for lipoxygenases from and coral were also published previously (31, 32, 34, 58, 171).

Crystallization experiments on the lipoxygenases were previously hampered by an inherent flexibility in the protein (see Chapter Three). The hypothesis for the basis of the flexibility is that the two domains can adopt numerous conformations in solution relative to 52 one another, ranging from closely associated compact structures to minimally associated extended conformations. The existence of this degree of flexibility was evident in small angle X-ray scattering measurements on several mammalian lipoxygenases (172-174). The effect was also important for leukocyte 5-lipoxygenase, for which crystallization was only realized with a version of the protein modified to stabilize a compact configuration (38).

The strategy adopted here was to truncate the N-terminal C2 β–barrel domain altogether, and investigate the crystallization behavior of the C-terminal catalytic domain. There were several reports on truncated lipoxygenases previously. The mini-LOX, a trypsin digested soybean LOX-1 had limited stability and was not crystallized (49). Also, C-terminal domains of rabbit 15-LOX and human platelet 12-lipoxygenase were successfully reported, but retained only limited activity (50, 173). 4.2 Results

4.2.1 Expression of 12-lipoxygenase catalytic domain

The cDNA of the C-terminal domain of PLL was amplified by polymerase chain reaction and then subcloned into E. coli for expression. This domain was successfully expressed using auto-induction techniques (150). The protein without His-tag was purified using two anion exchange columns (HiPrep QFF and Source 15Q). The purification of the His-tagged protein was completed by the combination of an IMAC column and an anion exchange column (Figure 4-1). This purified His-tagged 12-lipoxygenase catalytic domain retained catalytic activity and was reasonably stable with a specific activity of 1.55  0.38

μmol·mg-1·min-1 compared to 8.14  0.59 μmol·mg-1·min-1 for the full length protein (Figure

4-2). The C-terminal domain rate measurements exhibit a shorter lag phase and suicide inactivation, which are different from the behaviors of the native protein (Figure 4-2).

53 Figure 4-1 SDS-PAGE of HLCDS purification. Lane 1. Low MW standard markers; 2. Whole cell lysate; 3. Elution after Ni-NTA column; 4. Purified HLCDS after source column; 5. Purified PLL.

Figure 4-2 An example of the UV kinetics of lipoxygenase catalytic domain. AA concentration was 50 μM; HLCDS concentration was 150 nM.

54 4.2.2 Crystallization of 12-lipoxygenase catalytic domain

Crystallization experiments were conducted using three different constructions of leukocyte 12-lipoxygenase catalytic domain (LCD, LCDS, and HLCDS). Only HLCDS, the His-taged catalytic domain with two mutations (C210S/C292S), has shown the ability to produce crystals in the tested conditions. Thrombin cleavage of the His-tag from the protein led to failure of crystallization. The protein-OPP complex was prepared by the incubation of HLCDS with solid OPP at 4 oC for 16 hr. This complex was then subjected to crystal screening against several commercial crystallization kits. Initial crystals were found in Crystal Screen 2 #22 by the vapor diffusion method at room temperature (Figure 4-3).

Further optimization was carried on refining the concentration of precipitating reagent and adding a cryoprotectant. After the optimization, crystals were obtained from co-crystallization experiments using the hanging drop method in the condition of 5%~15%

PEG-20,000, 0.1 M MES buffer at pH 6.5, 20% glycerol at 18 oC (Figure 4.4.).

Figure 4-3 Initial crystal hit of HLCDS-OPP from crystal screen 2 #22. Crystallization condition: 11.7 mg·mL-1 HLCDS-OPP, 0.1 M MES pH 6.5, 15% PEG-20,000, vapor diffusion.

55 Figure 4-4 Crystals of HLCDS-OPP after optimization and addition of the cryoprotestant. Crystallization condition: 11.4 mg·mL-1 HLCDS-OPP, 0.1 M MES pH 6.5, 8% PEG-20,000, 20% glycerol, vapor diffusion.

4.2.3 Data collection and structure determination of HLCDS-OPP complex

Diffraction data of HLCDS-OPP crystals were first collected using a in-house X-ray source. However, the indexing of the data was hampered due to multiple reciprocal lattices in the diffraction images. Optimization of crystallization conditions and micro-seeding technique did not improve the diffraction quality of the crystals. In the synchrotron X-ray source, crystals of HLCDS-OPP were able to diffract beyond 1.6 Å with overlapping reflection lattices (Figure 4-5). With the application of a micro-focused beam collimator at

APS, several single crystal quality data sets were successfully obtained (Figure 4-5).

The structure was determined based on one data set of X-ray diffraction data extending to 1.89 Å by the molecular replacement method using the revised structure of

15-lipoxygenase (PDB ID 2P0M) as the search model in the CCP4 suite. Data collection and refinement statistics are reported in Table 4.1.

56 Figure 4-5 The diffraction patterns of HLCDS-OPP complexes using different size collimators. (A) In the pattern using 50 μm collimator, two reciprocal lattices from the image indicated the crystal mounted is not a single crystal; (B) In the pattern using 20 μm collimator, single crystal diffraction was obtained.

57 Table 4.1 Data collection and refinement statistics of HLCDS-OPP complex.

HLCDS-OPP complex (PDB 3RDE) Data Collection Space group P21 Cell dimensions a, b, c (Å) 83.5, 181.5, 91.6 α, β, γ (o) 90, 92.9, 90 Wavelength (Å) 1.0781 Resolution (Å) 48.97-1.89 (1.99-1.89) Rmerge 0.098 (0.284) I/σI 9.0 (3.8) Completeness (%) 99.5 (98.3) Redundancy (%) 3.6 (3.4) Wilson B factor 18.5 Solvent content (%): 54.0

Refinement Resolution (Å) 48.97-1.89 (1.94-1.89) No. reflections 214717 (15209) Rwork/Rfree 0.172/0.214 (0.213/0.252) No. atoms 18964 Protein/Solvent/Fe/Ligands 17580/1276/4/104 B-factors 22.4 Protein/solvent/Fe/Ligands 22.2/26.4/11.9/20.4 RMSD deviations Bond lengths (Å) 0.026 Bond angles (o) 1.974 Ramachandran Most Favored 1787 (92.3%) Additional Allowed 145 (7.5%) Generously Allowed 4 (0.2%) Disallowed 0

The values in parentheses are for the highest resolution shell; Rmerge   Ii (hkl)  Ii (hkl)  Fo Fc hkl i hkl = 100% ; R = 100% , where Fo and Fc are the  Ii (hkl)  Fo hkl i hkl observed and calculated structure factors, Rfree = test set 3.0%.

58 The structure of leukocyte 12-lipoxygenase catalytic domain was determined in the P21 space group containing four molecules in one asymmetric unit, arranged as a dimer of dimers through a two-fold axis (Figure 4-6).

Figure 4-6 Surface representation for one asymmetric unit of HLCDS-OPP complex. Two viewing angles of a cartoon representation are shown representing rotation by 75 degrees about a vertical axis. The colors of yellow, green, blue and red represent chain A, B, C and D, respectively.

Figure 4-7 Secondary structural features for 12-lipoxygenase catalytic domain. Two viewing angles of a cartoon representation are shown representing rotation by 90 degrees about a horizontal axis. The orange sphere indicates the position of the iron atom. Helix 2 is maroon.

The overall structure of lipoxygenase catalytic domain is primarily α-helical and contains the non-heme iron. This result indicates that the C-terminal domain of lipoxygenase is a structurally stable unit, while N-terminal β-barrel domain is an independent

59 domain for lipoxygenase regulation, having some structural contacts with the C-terminal domain. No obvious alterations and conformational changes in the catalytic domain could be attributed to the deletion of the N-terminus. The structure of the truncation protein LCD shares the secondary structure features of known mammalian lipoxygenases, except the variation in the position of helix α2 (Figure 4-7).

4.2.4 Specific inhibitor in the structure of 12-lipoxygenase catalytic domain

The isoenzyme specific inhibitor, OPP, was found in all four monomers in the asymmetric unit. The molecule of OPP was adjacent to the non-heme iron site where catalysis occurs. In the structure of the complex, OPP was associated with the catalytic domain of 12-lipoxygenase in a U-shaped channel that was open at one end to the surface of the protein (Figure 4-8).

Figure 4-8 The location of the OPP binding site in HLCDS-OPP complex. (A) A surface representation of the catalytic domain indicates the location of inhibitor. N-terminal domain modeled from the structure of 15-lipoxygenase is presented in green. (B) A cutaway surface representation shows the 2Fo-Fc map and the structure of OPP. The orange sphere indicates the position of the iron atom, while the red sphere for the iron-associated water/hydroxide. The 2Fo-Fc map was contoured at 1.5 .

The carboxylate of the inhibitor formed hydrogen bonding interactions with Gln-596 at

60 the opening of the channel (Figure 4-9). Arg-403, the putative ligand for the substrate (39), was found close by, but interaction with the inhibitor was mediated by three water molecules. In addition, the occupancies of the carboxylate groups for the OPPs were refined to be 0.5 suggesting that the orientation could be flexible except for the presence of this one dominant conformation. Otherwise, the contacts between OPP and the protein were all hydrophobic interactions. For example, Leu-179 and Leu-593 were located in the channel opening in a position to facilitate the penetration of the hydrophobic inhibitor.

Gly-407, and Leu-597 extended this hydrophobic interaction surface. Phe-175 was perpendicular to the aromatic ring of the inhibitor, and at a distance of 3.2 Å was in a position to form a CH-π-interaction. Ile-400, Leu-408, Ala-404, Ile-597, and Ile-663 were interacting with C10-C15 of OPP. The inhibitor binding site was terminated in a pocket of hydrophobic residues including Phe-353, Val-418, Val-419, Phe-415, and Ile-414. The triple bond of the inhibitor was adjacent to the non-heme site at a distance of roughly 4 Å from the iron. The metal-ligated water molecule was on the channel surface at a distance of

3.05 Å from the triple bond.

Figure 4-9 The specific interactions between OPP and HLCDS. A stereodiagram representing the amino acid side chains interacting with OPP.

61 4.2.5 Expression of recombinant manganese 12-lipoxygenase catalytic domain

To obtain the manganese recombinant 12-lipoxygenase catalytic domain, an external source of manganese was added into M9 minimal medium. The purification was completed by a combination of an IMAC column (Ni-NTA, Qiagen) and an anion exchange column

(Source15Q, GE Health Science). In the enzymatic assay, this purified recombinant

Mn-HLCDS displayed much less catalytic activity than the native form (Figure 4-10).

However, in the atomic absorption experiments, the iron content was determined as 9.4 ± 4.1

% while the manganese content was determined as 46.7 ± 0.2 %.

Figure 4-10 The kinetics profile of Mn-HLCDS. AA concentration was 50 μM; HLCDS concentration was 154 nM.

4.2.6 Crystallization of Mn-HLCDS-OPP and HLCDS–AA complexes

Crystals of the complex of Mn-HLCDS and OPP were produced by vapor diffusion using the same condition as HLCDS-OPP (Figure 4-11). For the co-crystallization of

HLCDS-AA, the employment of similar conditions failed to give any crystals. However, using small needle crystals of HLCDS-OPP as seeds, in some of crystallization experiments,

62 the protein would crystallize as small crystals with the longest dimension of approximately

0.1 mm (Figure 4-12).

Figure 4-11 Crystals of Mn-HLCDS-OPP complex. Crystallization condition: 12 mg·mL-1 Mn-HLCDS-OPP, 0.1 M MES pH 6.5, 10% PEG-20,000, 20% glycerol, vapor diffusion.

Figure 4-12 Crystals of HLCDS-AA complex. Crystallization condition: 11.7 mg·mL-1 HLCDS-AA, 0.1 M MES pH 6.5, 10% PEG-20,000, 20% glycerol; macro-seeding technique.

63 Table 4.2 Data collection and refinement statistics of Mn-HLCDS-OPP and HLCDS-AA complexes.

Mn-HLCDS-OPP HLCDS-AA Data Collection Space group P21 P21 Cell dimensions a, b, c (Å) 83.7, 182.3, 91.1 82.8, 182.6, 90.9 α, β, γ (o) 90, 92.9, 90 90, 93.2, 90 Wavelength (Å) 0.97856 0.97856 Resolution (Å) 39.14-1.92 (2.03-1.92) 42.49-2.50 (2.64-2.50) Rmerge 0.116 (0.604) 0.214 (1.533) I/σI 6.4 (2.9) 5.8 (1.5) Completeness (%) 94.1 (95.5) 100 (100) Redundancy (%) 3.0 (2.0) 4.3 (4.3) Wilson B factor 20.3 46.8 Solvent content (%) 53 52

Refinement Resolution (Å) 37.28-1.92 (1.97-1.92) 42.49-2.50 (2.59-2.50) No. reflections 194170 (14042) 92426 (9142) Rwork/Rfree 0.203/0.244 (0.284/0.324) 0.220/0.281 (0.283/0.365) No. of atoms (protein 17636/541/4/78 17644/358/4/na /solvent /Fe /ligands) B-factors (Protein 25.0/21.4/14.4/20.3 36.2/30.8/20.1/na /solvent /Fe /ligands) RMSD deviations Bond lengths (Å)/ 0.0245 Å /2.270o 0.0148 Å/1.453o angles (o) Ramachandran Most Favored/allowed/ 97.37/2.40/0.23 96.74/2.94/0.32 disallowed (%) The values in parentheses are for the highest resolution shell; Rmerge   Ii (hkl)  Ii (hkl)  Fo Fc hkl i hkl = 100% ; R = 100% , where Fo and Fc are the  Ii (hkl)  Fo hkl i hkl observed and calculated structure factors, Rfree = test set 3.0%; Fe was used in the model of Mn-HLCDS-OPP complex.

64 4.2.7 Data collection and structure determination of Mn-HLCDS-OPP and

HLCDS-AA complexes

The crystal diffraction data of Mn-HLCDS-OPP and HLCDS-AA complexes were obtained using a micro-focused beam (20 μm collimator) at the APS synchrotron X-ray source. Mn-HLCDS-OPP complex was able to diffract to 1.9 Å, while HLCDS-AA complex diffracted to 2.5 Å. The structural determination of these two complexes was carried out by the molecular replacement method using HLCDS-OPP complex as the model in the CCP4 suite (155). Iron (Fe) was used for the structure model of manganese recombinant protein since the manganese content of this protein is still low, and the atomic scattering factors of iron and manganese are very similar. Data collection and refinement values are summarized in Table 4.2.

4.2.8 An inhibitor unbound form of 12-lipoxygenase catalytic domain from the crystal structure of Mn-HLCDS-OPP complex

The overall structure of the Mn-HLCDS-OPP complex is almost the same as that of the

HLCDS-OPP complex. In one asymmetric unit, four monomers are assembled in the same way as that of the HLCDS-OPP complex. However, the electron densities corresponding to the molecule of OPP were found in three of these four monomers, while the fourth monomer only has very little electron density in the inhibitor binding site (Figure 4-13). The root mean square deviation (RMSD) between the unbound form of Mn-HLCDS-OPP and monomer A of HLCDS-OPP complex is as small as 0.322 Å (Figure 4-14), which is close to the value between any of two monomers in the HLCDS-OPP complex. This observation further reinforced the idea that the substrate binding pocket was pre-existing in the protein

65 instead of an induced fit model for enzyme-substrate interaction, which was proposed as the basis for the 15-lipoxygenase-inhibitor complex structure (37).

Figure 4-13 Alignment between the unbound form from Mn-HLCDS complex and the inhibitor bound form from HLCDS-OPP complex. Protein HLCDS in purple cartoon representation (chain A), and Mn-HLCDS in blue cartoon representation (chain D); the metal is presented as an orange sphere.

Figure 4-14 Apo lipoxygenase catalytic domain in the Mn-HLCDS-OPP complex. Fo-Fc map was contoured at 3.0 σ in green, 2Fo-Fc map contoured at 1.5 σ in blue. Fe is in orange; water in red; OPP molecule is modeled in balls and sticks.

66 4.2.9 The position of AA in HLCDS-AA complex

In the crystal structure of HLCDS-AA, four monomers were present in one asymmetric unit. The value of RMSD between monomer A of HLCDS-AA and monomer A of

HLCDS-OPP (all residues alignment) is 0.238 Å, indicating that no conformational alteration took place upon the addition of AA (Figure 4-15). However, there are no continuous electron densities in either Fo-Fc map or 2Fo-Fc map in the substrate pocket (Figure 4-16).

Only fragments of electron densities were found along the binding channel. One of the reasons could be that the substrate arachidonic acid could be converted into the hydroperoxides, which may be further converted into other products. Alternatively, the flexibility of arachidonic acid at both the alky tail and the carboxylic end could contribute to this observation.

Figure 4-15 Alignment of the HLCDS-OPP and HLCDS-AA complexes. The protein is presented in blue cartoon; Fe is in orange; water in red.

67 Figure 4-16 Electron densities in the active site of HLCDS-AA complex. Fo-Fc contoured at 3.0 σ in green, 2Fo-Fc contoured at 1.5 σ in blue. The protein is presented in semi-transparent tubes, Fe is in orange, water in red, and arachidonic acid molecule is modeled in balls and sticks.

4.3 Discussion

4.3.1 Crystallization conditions for 12-lipoxygenase catalytic domain

Two crystallization hits produced crystals in the crystal screening experiments. Except the one used for crystal preparation, the other condition was 1:1 ratio of 12 mg·mL-1

HLCDS-OPP complex with the kit solution containing 0.1 M Bis-Tris pH 5.5, 0.1 M ammonium acetate, 10% PEG-10,000. Crystallization and optimization of this condition failed to give diffraction quality crystals. Since the condition of 0.1 M MES pH 6.5, 5~15%

PEG-20,000, 20% glycerol gave beautiful crystals, the second condition was not further pursued. In the crystallization experiments, the inhibitor is a crucial component for the success in both conditions. In the same condition of crystallization, the protein without inhibitor will give oily drops instead of crystals. Seeding techniques (including micro- and

68 macro-seeding) were unsuccessful in preparation of the unbound form crystals. The temperature of the crystallization condition was not that critical, as crystals were grown at ambient room temperature, 18 oC, and 4 oC. For convenience, most of the crystallization experiments were carried out at 18 oC.

Of the three constructions of 12-lipoxygenase catalytic domain, only the one with a

His-tag yielded crystals in the crystal screening experiments. However, these N-terminal residues of the expression tag (including His-tag and thrombin cleavage site) were not present in the electron density map. If we performed a thrombin cleavage during or after the purification, the cleaved 12-lipoxygenase catalytic domain failed to form crystals under similar conditions. This indicates the N-terminal His-tag is important for crystallization.

Among the data in PDB, up to 40% of deposited proteins were crystallized with various expression tags (175). Sometimes, the expression tag may provide additional intermolecular interactions which facilitate crystal formation (175). Besides these, the

HLCDS was able to form crystals in the presence of inhibitors under similar conditions even if an in situ thrombin cleavage was applied.

4.3.2 Variation of helix α2 in mammalian lipoxygenases

The three-dimensional structure of the 12-lipoxygenase catalytic domain closely resembled the structures of leukocyte 5-lipoxygenase and rabbit 15-lipoxygenase (37, 38).

The secondary structural features were almost entirely shared with one conspicuous exception, the position and conformation of the amino acids that make up helix α2. A comparison of the mammalian lipoxygenases in this region of their structures is presented in

Figure 4-17.

69 Figure 4-17 A comparison of the positions of helix 2 in the structures of the mammalian lipoxygenases. The position of the HLCDS-OPP complex is in maroon, and the position of the iron atom is indicated by an orange sphere. Left: apo 15-lipoxygenase structure in light blue. Middle: 15-lipoxygenase-RS75091 complex in green. Right: 5-Lipoxygenase structure in blue.

The reevaluation of the original X-ray diffraction data for rabbit 15-lipoxygenase concluded that the crystals of the enzyme-inhibitor complex contained an open, apo form and a closed form with the inhibitor present near the non-heme iron site (37). The differences in the structures were primarily related to the position and conformation of the amino acids that make up helix α2. The position of this helix in the 12-lipoxygenase catalytic domain was much more similar to the location in the open, apo structure of 15-lipoxygenase than to the inhibitor bound closed form. The positions of these amino acids in the 5-lipoxygenase structure were quite distinctive. They were found in a short, three turn helix in a unique orientation flanked on both sides by extended loops (38). The orange spheres in the figures represent the positions of the iron atoms, illustrating that these differences in structure were in the portion of the protein covering the active site. The interpretation for the presence of two conformations in 15-lipoxygenase was an induced fit mechanism for inhibitor binding.

The new structure of the 12-lipoxygenase catalytic domain indicated that a conformational change of this kind was not an absolute requirement for inhibitor binding, because OPP was bound directly to the open conformation of the protein. The variations of helix α-2 among three related enzymes highlight an amazing way in which nature takes a similar overall shape,

70 and changes it in the important details.

4.3.3 The iron sites of mammalian lipoxygenases

The catalytic domain consisted primarily of α-helixes and contained the non-heme iron site (Figure 4-7). The redox active, non-heme iron atom in 12-lipoxygenase was coordinated by four (His-361, His-366, His-541, and His-545), the main chain carboxylate from the C-terminus (Ile-663), and one water/hydroxide in a pseudo-octahedral configuration. The coordinated water molecule was also stabilized by a hydrogen bond with the non-ligated oxygen atom of the terminal carboxylate. This additional bonding resulted in small B-factors for the water molecules (9, 12, 18, and 13 for the four chains A-D in the asymmetric unit) that were comparable in magnitude to the B-factors of their bonded iron atoms (9, 9, 17, and 12, respectively). The average iron-ligand distance in the crystal structure was 2.23  0.06 Å.

Figure 4-18 A comparison of the iron coordination environments for the mammalian lipoxygenases. From left to right are the iron sites from 5-LOX, 12-LOX and 15-LOX. Distances are in Å.

The iron site in the 12-lipoxygenase catalytic domain was more ordered than the iron sites in 5- or 15-lipoxygenase (Figure 4-18). For example, in 5-lipoxygenase a water molecule was not as tightly coordinated to the iron and was not in a position to be as effectively stabilized by hydrogen bonding to the C-terminal isoleucine, resulting in a distance of 3.6 Å away from the iron and with a B-factor of 36 (compared to 9, 12, 18, and 71 13 in our complex), in one of the monomers in the structural model (38). No water molecule at all was observed in the non-heme iron site of the second monomer in the

5-lipoxygenase structure, or in the structure of 15-lipoxygenase (37). Further, the iron binding site in 12-lipoxygenase had a distorted octahedral geometry, but significantly less distorted than in the previously reported structures. The average deviation from ideal octahedral geometry for the ligand-iron-ligand angles was 4.5° roughly half the value observed in other structures. The difference among the structures of the iron sites in the various lipoxygenases is quite striking. This trend reinforces the notion that flexibility or mobility of the iron and its coordination sphere represents an aspect of the structure that is relevant to catalysis (56).

4.3.4 The binding pockets of mammalian lipoxygenases

The inhibitors in the structures of 12- and 15- lipoxygenases adopted quite different orientations in their respective proteins. This was consistent with the fact that a significant conformational change was necessary to account for inhibitor binding to 15-lipoxygenase.

A comparison of the orientations of the two inhibitor molecules is provided in Figure 4-19.

Alignment of the two structures was conducted using secondary structural elements. The relative position of RS75091 in the 15-lipoxygenase structure is shown for comparison. In the structure of 15-lipoxygenase, the inhibitor occupied a large “boot-shaped” cavity that was not connected to the surface of the protein. OPP and RS75091 were both found close to the iron site, but no other similarities were evident. The common features of the inhibitors, the carboxylate groups, aromatic rings, and hydrophobic tails were all found in different places in the two structures. The geometry enforced by the ortho-substitution pattern in RS75091 in all likelihood accounts for the different modes of binding. For example, this feature

72 would prevent entry to the U-shaped channel by direct insertion. The structure of inhibitor bound 15-lipoxygenase has been used as a starting point in molecular modeling and docking studies (176). The structure of the 12-lipoxygenase complex will also provide a template for such studies. Further, the results with 12-lipoxygenase and OPP indicated that there may be a structural basis for distinguishing between isoenzymes with inhibitors.

Figure 4-19 A comparison of the positions of OPP in the complex with 12-lipoxygenase and RS75091 in the complex with 15-lipoxygenase. The cutaway surface of 12-lipoxygenase and 15-lipoxygenase are in yellow surface representation in panels A and B, respectively. Iron and its coordinated water are presented in orange and red spheres, respectively. The inhibitors OPP and RS75091 are shown in sticks and balls, in purple and slate colors, respectively.

4.3.5 Implication for inhibition and enzymatic catalysis

OPP binds to both the ferrous and ferric forms of leukocyte 12-lipoxygenase, with a higher affinity for the ferrous form (Ki Fe(II) = 70 nM; Ki Fe(III) = 2000 nM) (152). The tight-binding to the ferrous form accounts for the inhibition mechanism, which is preventing activation of the enzyme by hydroperoxide-dependent oxidation of ferrous inactive to the ferric active enzyme. This implies that the U-shaped channel is also the site of fatty acid

73 hydroperoxide binding because iron oxidation requires direct contact between the metal center and the hydroperoxide group.

Examination of the structure of the HLCDS-OPP complex reveals that the alkyne in the molecule of OPP is positioned immediately adjacent to the non-heme iron. The rigidity of the alkynl group in OPP is preventing the activation and hydrogen abstraction of leukocyte

12-lipoxygenase. The phenylpropionate mimics the carboxyl group and double bond at the

C5 position of arachidonic acid, while the long-chain alkyl groups provide similar hydrophobic properties as the other end of AA. These observations are consistent with the proposed mechanism of inhibition (152).

Arachidonic acid fit well into the electron density for OPP in the 2Fo-Fc map, indicating that the U-shaped channel could serve as the substrate binding site. The position of arachidonic acid identified in this way is illustrated in Figure 4-20. In this model, there is also an extended cavity from the non-heme iron site to the surface of the protein that intersects the substrate channel, providing the dioxygen channel necessary for lipoxygenase catalysis (Figure 4-21). The pathway displays amphiphilic features with a few hydrophilic residues on one side of the openning of the cavity, while the end nearest to iron is exclusively occupied by hydrophobic residues. This gradient of hydrophobicity may facilitate the movement of dioxygen along the channel to the substrate. In the bottom of the channel, hydrophobic residues, such as Leu-362, and Leu-367 may play critical roles in directing the movement of dioxygen. An increased Michaelis constant for oxygen was observed in the case of the L367F mutant of rabbit 15-lipoxygenase (177). A single amino acid, alanine critically influences the regio- and stereo-specificity of the peroxygenation reaction according to site-directed mutagenesis experiments carried out on 15S-lipoxygenase (62).

74 The side chain of Ala-404 in PLL impeded dioxygen targeting of the C8 position of AA, enforcing the S-stereochemistry (Figure 4-21). The orientation of the arachidonic acid molecule and the position of O2 pathway in the structural model (Figure 4-21) verifies the hypothesis for the stereochemical outcome for the catalyzed reaction.

Figure 4-20 Modeled arachidonic acid in the U shaped channel. Arachidonic acid was modeled into OPP’s 2Fo-Fc electron density map, and Fe and the coordinated water were from the structure of HLCDS-OPP complex.

Figure 4-21 Stereodiagram of the dioxygen pathway in the 12-lipoxygenase catalytic domain. The dioxygen pathway computed with a probe radius of 1.2 Å by Hollow 1.2 (178) is represented in light blue voids. Arachidonic acid is colored in gray in sticks and balls. Fe is shown in orange sphere, while water is in red.

75 In this model, one of the hydrogens attached to C10 was the closest substrate hydrogen to the Fe-OH complex with an H to O distance of 1.7 Å (Figure 4-20). The orientation of the arachidonic acid molecule in the model was also consistent with the stereochemical outcome for the catalyzed reaction. If hydrogen atom abstraction at C10 took place from the back to be consistent with the location of the FeOH, and the oxygen molecule reacted at

C12 from the front as arachidonic acid is oriented in the figure, the result would be the formation of the observed 12S-hydroperoxide (Figure 4-22).

R1 HR R1 HS HR 8R Fe-OH C8 C8 O2

R2 R2 12S C12 C12 O2

Figure 4-22 Implication for lipoxygenase catalysis. Arrows indicate the abstraction of hydrogen, and the positions of addition of dioxygen (35).

4.3.6 Substrate/intermediate binding site in 12-lipoxygenase

The crystal structures of Mn-HLCDS-OPP and HLCDS-AA complexes revealed more details of the substrate binding site in leukocyte 12-lipoxygenase. There is an apo structure of the catalytic domain observed in the Mn-HLCDS-OPP complex with high fidelity to the

HLCDS-OPP structure. This structure confirms that there are minimal conformational changes between the unbound form and the inhibitor bound form. However, this observation does not exclude the possibility that the protein may experience conformational changes to facilitate the entering of the substrate or inhibitor. Since this U-shaped channel is pre-existing in the unbound enzyme, the structural fluctuation upon this interaction should

76 be minimal only involving some local residues instead of global movements. Alternatively, the substrate may penetrate the channel using a tail-first orientation without disrupting the preformed substrate channel.

From the crystal structure of HLCDS-AA, some electron density was found along the substrate channel. However, the position of AA is not as fixed as the inhibitor OPP in the complex, which is normal for a flexible molecule. Different products, for instance,

12-HPETE, could be present in the crystallization drop due to the reactivity of the enzyme.

For the future, co-crystallization experiments with 12-HPETE or 15-HPETE should be carried out as the product-enzyme complex would be informative for the detailed understanding of catalysis. Also, crystallization experiments performed under anaerobic conditions may lead to the formation of the substrate enzyme complex since the enzymatic activity should be completely blocked due to the lacking of the co-substrate, dioxygen. The mutagenesis studies of some key residues along the substrate channel will possibly lead to capturing the substrate protein complex in crystallization experiments and revealing some details about the interaction between lipoxygenase and arachidonic acid. As mutants containing one or a few key residues (Figure 4-21) in the dioxygen pathway may not have the enzymatic activity, another strategy to obtain the substrate-enzyme complex is the co-crystallization of one of these mutants and arachidonic acid.

The in vitro metal replacement experiment may be another way to capture to the substrate-protein complex, which has been successfully used in cyclooxygenase-substrate complexes (179) and many other metaloproteins. However, this method would be limited to protein that can withstand metal removal and its replacement under partially denaturing conditions. The expression and purification of metal replaced heme protein has been

77 reported in E. coli (180). Our preliminary recombinant metal substitution experiments described in this dissertation indicated that E. coli have the ability to take up exogenous metal from the expression medium for the assembly of recombinant even though it is unclear how the iron and other metals are delivered. The ingredients of the medium, the expression conditions, and the choice of metals could be further optimized for the expression of protein with non-native metals.

78 Chapter 5

Conclusions

This dissertation describes the biochemical characterization of porcine leukocyte

12-lipoxygenase, and the crystal structure of the catalytic domain. Lipoxygenases are a class of non-heme, non-sulfur iron dioxygenasess that catalyze the incorporation of molecular oxygen into polyunsaturated fatty acids to give hydroperoxides. The products from lipoxygenase catalysis are intermediates in the formation of bioregulators responsible for the modulation of basic physiologic processes such as inflammation, fever, blood-clotting, and hypersensitivity reactions. Understanding of 12-lipoxygenases is important, for example, because bioactive lipids from leukocyte 12-lipoxygenase have anticarcinogenic effects while those from the platelet isoform could be involved in procarcinogenic effects.

Full length porcine leukocyte 12-lipoxygenase was expressed and purified using modern cloning techniques. In electrospray ionization mass spectroscopic measurements, the loss of the mass of an iron atom from the protein as the pH declined from 5 to 4 for native leukocyte

12-lipoxygenase was observed. During this acid-induced unfolding process, the distribution of protein ionization states was not changed compared to the native form. This observation was in stark contrast to the behavior of similarly treated soybean LOX-1. This result indicates that the iron site of leukocyte 12-lipoxygenase behaves differently from other

79 lipoxygenases and this acidic induction method may be useful to obtain the apo form of this enzyme.

Extensive crystallization experiments were conducted to obtain crystals of leukocyte

12-lipoxygenase using sparse matrix screening kits. Many site-directed mutagenesis experiments and multiple expression protocols were explored. Only one crystal hit was found for the full length protein and it did not diffract well in X-ray diffraction experiments.

Global site-directed mutagenesis was also employed to increase crystallizability of leukocyte

12-lipoxygenase. Two cysteines (Cys-210 and Cys-292) that were found exposed on the surface of the model of leukocyte 12-lipoxygenase were mutated into serines to enhance the half-life of leukocyte 12-lipoxygenase in concentrated solutions. Also mutagenesis was applied to mutate three clusters (45-46, 266-268, and 611-613) of residues suggested by

Surface Entropy Reduction prediction to alanines to reduce the entropy barrier during crystallization. However, these mutants failed to produce crystals in the crystal screening experiments.

The C-terminal catalytic domain of 12-lipoxygenase was constructed using molecular cloning techniques and expressed in E. coli. This domain was able to crystallize as a complex with its inhibitor 4-(2-oxapentadeca-4-yne) phenylpropanoic acid (OPP). The crystal diffracted to 1.89 Å in the P21 space group using a micro-focused beam at the

Advanced Photon Source. The structure was determined by molecular replacement with

R-work 17.2% and R-free 21.4% (PDB id: 3RDE). The C-terminal domain is primarily

α-helical and contains the non-heme iron. It shares the same folding of known mammalian lipoxygenase structures, except the variation of the position of helix α2. This new structure of the 12-lipoxygenase catalytic domain with OPP bound revealed a new one-open-end

80 U-shaped channel for the natural substrate arachidonic acid, which is remarkably different from the inhibitor (RS75091) binding pocket of rabbit 15-lipoxygenase. This structure and the new proposed channel account for the regio-/stereo- specificities of catalysis, and will pave the road for the discovery of new inhibitors for mammalian lipoxygenases.

To reveal the details about the natural substrate binding pocket of 12-lipoxygenase, crystallization of the 12-lipoxygenase catalytic domain with arachidonic acid was also performed. A macro-seeding technique was applied to obtain the complex of

12-lipoxygenase catalytic domain and arachidonic acid. In a 2.5 Å resolution crystal structure, the whole molecule of arachidonic acid did not fit well in the Fo-Fc map in the U shaped channel. However, fragments of electron densities were found along the binding channel indicating that arachidonic acid was not fixed well in the site or it was converted into hydroperoxides and other derivates. The structures of the two complexes were nearly identical suggesting that OPP binds to the protein in the same way as the substrate arachidonic acid.

In addition, a recombinant manganese reconstituted lipoxygenase catalytic domain protein was expressed, but the Mn content was only about 50%. This manganese enzyme exhibited little activity, maybe due to the presence of some iron left in the protein (iron content was about 10%). This protein was crystallized using the same condition as the native iron form. A 1.9 Å structure of the manganese catalytic domain-inhibitor complex suggests that three monomers out of four in the asymmetric unit have OPP bound in the same manner as that of the native form. In the fourth monomer of the asymmetric unit, the molecule of OPP was absent. This observation reinforced the idea that there are no structure fluctuations or conformational changes upon the binding of OPP to the catalytic

81 domain. Further expression studies of the manganese enzyme should provide new methodology to prepare metal substituted protein in the E. coli expression system and extend the enzyme-substrate complex studies.

In summary, this research resulted in detailed biochemical studies of recombinant porcine leukocyte 12-lipoxygenase and its catalytic domain. The crystal structure of

12-lipoxygenase catalytic domain revealed new structural features of this enzyme. The structure of enzyme-inhibitor complex should assist drug design and also facilitate further characterization of mammalian lipoxygenases.

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