Insights Into 5-Lipoxygenase Active Site and Catalysis Sunayana Mitra Louisiana State University and Agricultural and Mechanical College

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2015 Insights into 5-Lipoxygenase Active Site and Catalysis Sunayana Mitra Louisiana State University and Agricultural and Mechanical College

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This Dissertation is brought to you for free and open access by the Graduate School at LSU Digital Commons. It has been accepted for inclusion in LSU Doctoral Dissertations by an authorized graduate school editor of LSU Digital Commons. For more information, please [email protected]. INSIGHTS INTO THE 5- LIPOXYGENASE ACTIVE SITE AND CATALYSIS

A Dissertation

Submitted to the Graduate Faculty of the Louisiana State University and Agricultural and Mechanical College in partial fulfillment of the requirements for the degree of Doctor of Philosophy

The Department of Biological Sciences

by Sunayana Mitra B.S., Nagpur University, 2005 M.S., Nagpur University, 2007 August 2015

Dedicated to Ma and Baba, (Mom and Dad)

कर्मणयेवाधिकारते र्ा फलेष ु कदाचन। र्ा कर्मफलहेतर्ु मर्ामभ ते सङ्गोऽ्वकर्मधण। (2.47, the Bhagvad Geeta) (Do your duties without thinking of gains or losses, let not the fruits of labor be your motivation and let not be faulted of not doing)

iii ACKNOWLEDGEMENTS

Ever since I was a little kid I have always wanted to become a scientist. Even though back then I had perhaps only a very vague idea of what that really entails. I think it was fed into my head by mother, even though completely unintentionally, when she used to call me her

‘scientist daughter’. And the reason for that was my unending fascination with screwdrivers and the things you could unscrew with them! I remember opening up every last toy I received that had even one screw to see how it worked and what went inside. Of course my lack of expertise in such matters meant that I had a basket full of toys which I could never put back together in working condition. So firstly I would like to thank that person who taught me how a screw driver works at age 3. I am not sure if it was my father or someone else, but that really fueled my inquisitiveness which is still going pretty strong.

I have also being really lucky to have some wonderful teachers during the years, without whom I would not have found my way and reached this point here. The very first name that comes into my mind is Mrs. Bhattacharya who inspired me to be confident while speaking in

English. I am still indebted to her for giving me the gift of language. I would also like to that

Ms.Rita Poothia, who always inspired me to think out of the box and outside textbooks. I would probably left biology out completely after my tenth grade to focus on my love of physics. But by the twelfth grade I realized that Biology is after all what I really love. I would also like to thank friends from school, who have still kept touch with me and are always concerned about my wellbeing. Thank you Mr. Santosh K. Dash and Dr. Suchismita Datta for being there, always.

Moving on to my undergraduate years, I am highly indebted to Dr. Kakoli Upadhyay for instilling in me the passion for science and research. She has been instrumental in steering me to major in Biochemistry and Chemistry. I am also extremely thankful to Dr. Avinash Upadhyay,

iv my mentor and advisor during my Masters’, to inspire me to think objectively and not rely solely on bookish knowledge alone even when it comes to understanding fundamentals in Science and question everything! He was always there to patiently answer my unending questions about pretty much every topic that was ever covered during those two years. Academics apart his guidance and support to me in times good and bad when I was all by myself in an unfamiliar place. Thank you Kakoli Ma’am and Avinash Sir for being there for me.

When you are extremely shy and introverted and find yourself in a new city miles away from home and you are all by yourself for the first time in life, friends become the only family that you can count one. I can’t thank enough all the roommates from different hostels, I had a chance to share my life with during my years Undergrad and Masters’ in Nagpur City. They made it possible for me to live without seeing my family for years together at time. Thank you

Ms Sayma Khan for being the awesome friend you are. I also want to thank Ms Nishigandha

Naidu, Ms Kanta Upadyaya, Ms Divya Chowdhary and everybody else who made it a little easier to live independently. I think that the experiences are helping me even now that I so far away.

I also was fortunate enough to make some wonderful friends during the same period who have been extremely kind and caring. Thank you Mr. Robin Asati and Dr, Tanushree Pandit for being the most wonderful friend anyone could ever ask for. Thank you guys for putting up with me, in spite of all my eccentricities and quirks, and sticking with me through thick and thin. I can’t imagine my life without you guys.

I came to LSU in fall 2008 to begin my stint as a graduate student in the Biochemistry

PhD program. Again being in completely alien surrounding, I could not have managed without the kind help people gave right from the moment I arrived all the way from India. Thank you so

v much Dr. Sreerupa Ray for being the most helpful and kindest roommate I could have ever asked for. I also would like to thank Dr. Sona Chowdhury, Mr Pratik Dhar and Ms Sanchita Dhar for being kind enough to help me set up my life here in Baton Rouge.

Even though things did not work out as planned, I was given another chance at it due to the extreme kindness of Dr. Marcia Newcomer. I will always be indebted to her for her acceptance of me in her lab without any hesitation. She has always meticulously guided me through the nitty gritty of protein research, a hitherto unfamiliar subject for me. I am also highly appreciative of her immense patience in working with me. I know I am not an easy person to work with and I thankful to her she for looking past my transgressions and steering me in the right direction nonetheless. I would also like to thank my committee members Dr. Anne Grove and Dr. Diana Williams for their valuable advice and support. Dr. Williams also generously allowed me to utilize the facilities at the Hansen’s disease Center to train in molecular techniques. Her kind words of encouragement have always inspired me to not give up. I would like to thank Dr. Frank Andrews for his valuable time in participating as the Dean’s representative.

I would like to thank Dr. Nathaniel Gilbert for getting me started in the lab. I would also like to thank Dr. Günes Bender for being a wonderful friend and guide inside and outside the lab.

I would like to thank Dr. Vijai Krishnan for being my friend, philosopher, guide and so much more. You have been my rock though the years and I hope I have your support in future as well. I don’t think I would have managed to finish the program without you. Thank you Vijai for holding my hand through the tough times and thank you for your gift of humor that always helps cut through all my intensity.

I would also like to thank my other pillar of support, my mother Ms. Susmita Mitra. She has not only single handedly raised me and my sister without complaints, but has always being there for me whenever whether near or far. Thank you Ma for letting me follow my heart in spite of our difficult life situations. I really do hope Ma that I have not disappointed you too much.

I would also like to thank my sister Ms. Suhasini Mitra, who although years younger to me, has always been much more worldly wise. She has always had valuable advice for her naïve older sister. I would like to thank my cousin brother Mr. Sudipta Mitra for always being ready with all kinds help and advice whenever I needed them. And I would like to also thank my Uncle

Mr. Anjan Sen and Aunt Mrs Sucheta Sen for their love and support. I would also like to thank my late grandmother Ms Atasi Dey for being the ideal I will always look up to. For a women born in her day and age she was not only college graduate majoring in mathematics, she was also heavily involved in organizations aimed at training and rehabilitation of oppressed and abused women in her adult life. Dida you will always be an inspiration.

Finally this section would be incomplete without thanking my late father. Even though we got spent little time together Baba, I will cherish those fond memories for lifetime. Thank you for all the love and affection you had for me. Thank you for always fulfilling my every wish big and small. An even though you are no longer physically here with me, thank you for always guiding me in spirit. I really hope, that at least partially, I have become the woman you wanted me to become.

vii TABLE OF CONTENTS

ACKNOWLEDGEMENTS…………………………………………………….…………. iv

LIST OF TABLES…...………………………………………………………………….... ix

LIST OF FIGURES……...... …………………………………………………………….... x

ABSTRACT……………………………………………………………………………..... xii

CHAPTER 1: INTRODUCTION...... 1 1.1 The Lipoxygenase Enzyme Family: A Structural Overview...... 1 1.2 Mammalian Lipoxygenases and their Biological importance...... 4 1.3 Leukotrienes: Pathophysological Importance...... 7 1.4 Human 5- Lipoxygenase - relationship between structure and function...... 10

CHAPTER 2: IDENTIFICATION OF THE SUBSTRATE ACCESS PORTAL OF 5- LIPOXYGENASE...... 15 2.1 Overview...... 15 2.2 Introduction...... 16 2.3 Results...... 22 2.4 Discussion...... 35 2.5 Materials and Methods...... 40

CHAPTER 3: CONCLUSIONS...... 45

REFERENCES...... 49

VITA...... 60

viii LIST OF TABLES

1. Human ALOX genes and major expression sites of the corresponding LOX-isoforms…...... 5

2. Utilization of substrate analogs by ‘trimmed’ mutants……………………………….……… 27

3. Utilization of substrate analogs by ‘trimmed’ mutants………………………………….…... 28

4. a) Summary of Thermal Shift and Enzyme Kinetics measurements of ‘plug’ mutants…….. 31 b) Summary of product formation by the ‘plug’ mutants……………………………….…... 31

5. Summary of different properties of the His600 mutants……………………………………. 34

ix LIST OF FIGURES

1. General mechanism of Lipoxygenase reaction…………………………………………… 2

2. Lipoxygenase structure comparison…………………………………………………….... 4

3. Biological functions of Lipoxygenases……………………….………………………….. 7

4. Leukotriene Synthesis Cascade……………………………………………………….….. 9

5. Comparison of α2 helix of 5-LOX with other LOXs…………………………..…….…... 12

6. Coordination of Catalytic Fe in 5-LOX …………..……………………………….…….. 12

7. The various states of catalytic Fe during 5-LOX action………………………………..... 13

8. Possible points of substrate entry in 5-LOX………………………………………...……. 15

9. Arachidonic Acid as a substrate……………………………………………………..….... 17

10. LOX product stereo-chemistries are consistent with one of two orientations of AA is the active site………………………………………………………………………………... 18

11. Schematic of HPLC analysis and sample chromatograms……………………….....….. 20

12. Comparison of AA and substrate analogs……………………………………...…..…... 21

13. Amino acids in Stable 5-LOX targeted for Site-Directed Mutagenesis……………..…. 22

14. Stable-5-lipoxygenase has an encapsulated active site…………….……………….…... 23

15. Measurements of Enzyme Kinetics………………………………………………….…. 24

x 16. A structural schematic of trimming the ‘plug’…………………………………………. 25

17. Effects of trimming the ‘plug’ on enzyme activity……………………………………... 25

18. Effect of trimming the ‘plug’ on 5-LOX structural and functional integrity………….... 26

19. Utilization of 5-HETE as a substrate by ‘plug’ mutants…………………………….….. 27

20. Effect of unplugging the ‘plug’ on 5-LOX activity……………………………….……. 28

21. Effect of unplugging the ‘plug’ on 5-LOX structural and functional integrity…..…….. 29

22. HPLC traces of unplugged mutants……………………………………………………. 30

23. W147L mutant destabilizes the enzyme both structurally and functionally…………… 31

24. Position of His600 in the enzyme active site…………………………………………... 32

25. Effect of His600 mutants on Thermal Shift and Enzyme Kinetics measurements…...... 33

26. Product formation by the triple mutant as compared to stable 5-LOX……………....… 33

27. Displacement of the corking residues in 5-LOX…………………….……………..…... 39

28. Thermal Shift Assay (TSA) procedure………………………………………………..... 44

29. Position of His 600 at Enzyme Active Site…………………………………….……..... 46

30. Product assays of His432 mutants…………………………………...... ……….....… 47

xi ABSTRACT

Leukotrienes are potent immune-modulatory compounds involved in the inflammatory response. 5-Lipoxygenase (5-LOX) is the enzyme that catalyzes the production of Leukotriene

A4 (LTA4), which is further metabolized to pro- and anti-inflammatory compounds derived from the substrate Arachidonic Acid (AA), a polyunsaturated fatty acid. The crystal structure of a stabilized form of 5-LOX has been recently reported (Gilbert, Bartlett et al. 2011).

However, the structure does not reveal the mode of substrate entry or recognition.

The first step in the enzymatic reaction involves the removal of a hydrogen from the central carbon of a pentadiene present in the substrate. It is not known how 5-LOX distinguishes among three such chemically equivalent pentadienes in AA to produce only 5-S-

HPETE, which is subsequently transformed to LTA4. The crystal structure suggests a somewhat crescent shaped active site lined with invariant Leucines and Isoleucines, with no clear access port visible for substrate entry.

A series of site-directed mutants were produced and the activities of these mutants evaluated. Product analyses of these mutants with AA and substrate analogs suggest entry of the substrate by the opening of the so-called Phenylalanine (F)-Tyrosine(Y) ‘cork’. The data is also consistent with the orientation of AA as carboxyl innermost, as predicted from the stereochemistry of the product and mechanistic models of lipoxygenase function.

xii

CHAPTER 1. INTRODUCTION

1.1 The Lipoxygenase Enzyme Family: A Structural Overview

Lipoxygenases (LOXs) are a class of enzymes that produce hydroperoxy fatty acid precursors by addition of a molecule of oxygen onto a polyunsaturated fatty acid substrate containing at least one cis,cis-1,4-pentadiene moiety (Brash 1999, Andreou and Feussner

2009). Lipoxygenases have a widespread presence in the living world, being found in animal, plants, fungi and even bacteria (Joo and Oh 2012). Apart from that, multiple isoforms of lipoxygenase are generally present in a particular species having functional differences between them. For example, more than twenty different LOX genes have been recognized in the rice genome, seven different ones are seen in the murine genome and the human genome contains six functional LOX genes (Ivanov, Heydeck et al. 2010). The hydroperoxy fatty acid products generated by LOX action are further metabolized into bioactive compounds with diverse roles such as leukotrienes and lipoxins in higher animals (Haeggstrom and Funk 2011), jasmonic acids in plants (Mosblech, Feussner et al. 2009), and lactones in microorganisms

(Huang and Schwab 2011).

When we look at the basic fatty acid oxygenation by LOXs, it fundamentally consists of four sterically controlled reaction steps mediated by a non heme iron moiety: hydrogen abstraction, radical rearrangement, oxygen insertion and peroxy radical reduction (Ivanov,

Heydeck et al. 2010). (Figure 1) (Next page)

Figure 1: General mechanism of lipoxygenase reaction. The reaction is initiated by stereo selective hydrogen abstraction at the central carbon (Cn) of a cis, cis-1, 4-pentadiene; the resulting radical can migrate over the neighboring double bond at either Cn+2 or Cn-2 and react with molecular oxygen to form a peroxy radical. The oxidation of the active site iron provides an electron for the formation of a peroxyanion, which is then protonated into a hydroperoxide. The hydrogen abstraction and insertion of oxygen are anatarafarcial in nature i.e., they occur on opposite sides of the substrate.

Structurally, all lipoxygenases, irrespective of their origins, share a predominantly α- helical catalytic domain. The main feature of this domain is the highly conserved ‘catalytic core’ comprised of about 17 helices encompassing the catalytic iron within. The catalytic site is defined by four core helices, with helices α7 and α14 on one side of the iron moiety and the α8 helix (‘arched helix’) and the penultimate helix on the other side (Newcomer and Brash 2015).

The iron coordination sphere exhibits pseudo octahedral geometry with several invariant histidine side chains from the core helices α7 and α14 as well as the main chain carboxyl of the

C-terminal, invariant Ile, acting as the ligands (Gaffney 1996). The arched helix, so called due to its distinctive curvature found in all solved LOX structures, helps shield a U-shaped catalytic cavity from the outer solution state (Minor 1996).

In addition to the above, the animal and plant enzyme also have an additional β-barrel domain at the N-terminus, known as the Polycystin-1, Lipoxygenase, Alpha-Toxin (PLAT) domain. Even though the PLAT domains display a high degree of structural conservation, unlike the catalytic domain, they show much less sequence conservation. This domain of LOXs is involved in membrane binding aided by Ca2+ (Hammarberg, Provost et al. 2000, Kulkarni,

Das et al. 2002). This domain might also have a regulatory role in catalysis even though it is not vital for enzyme action (Ivanov, Di Venere et al. 2011). (Figure 2) (Next page). As the figures suggest it is also notable that the PLAT domain in plants is larger than animals. Also, the absence of a PLAT domain in bacterial LOXs is consistent with the fact that they are secreted as soluble enzymes (Hansen, Garreta et al. 2013).

Figure 2: Lipoxygenase Structure Comparison. Lipoxygenase structure examples from Bacteria (cyan), Plant (green) and Animal (magenta) are shown. The additional PLAT domains in Plant and Animal specimens are indicated by blue boxed regions.

1.2 Mammalian Lipoxygenases and their Biological importance

I will now focus on the mammalian LOX isoforms in general and 5-LOX in particular for their relevance to this body of work. The physiological importance of LOXs in the mammalian systems is highlighted by the fact that several isoforms have been discovered with diverse roles in maintenance of proper cellular function. Table 1 lists a summary of LOX isoforms present in human and their murine counterparts. summarizes and assigns names of the

4 genes to the different isozymes for the ease of identification of their respective activities (small letters for mouse and capital letters for human genes) (Kuhn, Banthiya et al. 2015).

Table 1. Human ALOX genes and major expression sites of the corresponding LOX-isoforms.

Human Former Mouse Former Major References gene name gene name expression ALOX5 5-LOX alox5 alox5 Leukocytes, (Funk, Hoshiko et al. macrophages, 1989, Ghosh 2003, dendritic cells Faronato, Muzzonigro et al. 2007, Wasilewicz, Kolodziej et al. 2010) ALOX12 pl12- alox12 pl12- Thrombocytes, (Funk, Furci et al. LOXb LOX skin 1990, Johnson, Brass et al. 1998, Virmani, Johnson et al. 2001) ALOX12B 12R- alox12b 12R- Skin (Boeglin, Kim et al. LOX LOX 1998, Meruvu, Walther et al. 2005) ALOX15 12/15- alox15 Lc12- Eosinophils, (Sigal, Grunberger et LOX LOXa bronchial al. 1988, Sigal, Sloane epithelium et al. 1993) ALOX15B 15- alox15b 8-LOX Hair roots, (Brash, Boeglin et al. LOX2 skin, prostate 1997, Jisaka, Kim et al. 1997) ALOXE3 eLOX3 aloxe3 eLOX3 Skin (Kinzig, Heidt et al. 1999, Yu, Schneider et al. 2006, Krieg and Fuerstenberger 2014) Pseudogene alox12e elox12 Skin (Funk, Chen et al. 2002) a: lc — leukocyte-type. b: pl — platelet-type.

Now, it is well known that LOXs are involved in modulating cellular activity by the formation of lipid mediators using free polyenoic fatty acid substrates. The primary products of the LOX pathway are subsequently converted to a large array of bioactive lipid mediators, which include leukotrienes (Savari, Vinnakota et al. 2014), lipoxins (Romano 2010),

5 hepoxilins (Pace-Asciak 2009), eoxins (Sachs-Olsen, Sanak et al. 2010), resolvins (Yoo, Lim et al. 2013), protectins (Serhan and Petasis 2011) and others originating from AA, EPA and

DHA. These signaling molecules have been seen in both the pro and anti- inflammatory responses. Apart from this, LOXs might affect cellular metabolism by other ways as well

(Figure 3).

Firstly, several LOX isoforms have been shown to be capable of oxidizing complex ester lipids (Belkner, Wiesner et al. 1991) and even lipid-protein assemblies such as cell membranes and lipoproteins (Schewe, Rapoport et al. 1986). The introduction of a hydrophilic peroxide group into the hydrophobic tail of a fatty acid changes the physiological properties of the ester lipids. Such oxidized lipids can come together to form “hydrophilic pores” within the lipid bilayer of a cell membrane. This impairs barrier function of the membrane which, in turn, may lead to cellular dysfunction(van Leyen, Duvoisin et al. 1998). This particular ability can also implicate LOXs in having at least a partial role in erythropoiesis (Koury, Sawyer et al. 2002), epidermal differentiation(Epp, Fuerstenberger et al. 2007, Kypriotou, Huber et al. 2012) and atherogenesis (Zhao and Funk 2004, Poeckel and Funk 2010).

Another way by which LOXs exert their activity is by forming lipid peroxides, LOXs can modify the cellular redox state. The cellular redox equilibrium is an important regulator of gene expression (Forman, Ursini et al. 2014) and such intracellular LOX’s activity has also being associated with cell proliferation and carcinogenesis (Nie, Hillman et al. 1998, Kelavkar, Nixon et al. 2001, DuBois 2003). Although the exact nature of their role remains unclear, the expression of LOXs in humans is highly affected in prostrate (Matsuyama and Yoshimura 2008) and colorectal (Cathcart, Lysaght et al. 2011) carcinomas.

Figure 3. Biological function of lipoxygenases. Lipoxygenases may exhibit their biological functionality via three different mechanistic scenarios: i) Formation of bioactive lipid mediators, ii) structural modification of complex lipid–protein assemblies and iii) modification of the cellular redox homeostasis, which alters the gene expression pattern(Kuhn, Banthiya et al. 2015).

1.3 Leukotrienes: Pathophysiological Importance

Leukotrienes are extremely pathologically significant, particularly due to their role in inflammation. As the name implies, leukotrienes are predominantly produced by leukocytes due to their high level of 5-LOX and FLAP expression. However, different cell types have different levels of actual LTs produced (Peters-Golden and Henderson 2007). Neutrophils

7 produce only LTB4 and eosinophils, mast cells, and basophils produce mostly cysLTs. In contrast, macrophages and dendritic cells are capable of making both LTB4 and cysLTs.

Certain non-leukocyte cell types in humans, such as bronchial fibroblasts and epithelial cells are capable of producing minor amounts of LTs owing to low level expression of 5-LOX and

FLAP (James, Penrose et al. 2006, Jame, Lackie et al. 2007). Overall tissue biosynthesis of

LTs is also affected by non-leukocyte cells due to their expression of distal LTA4 - metabolizing enzymes LTA4H and LTC4S. This enables them to produce LTs even in the absence of 5-LOX/FLAP through “transcellular biosynthesis” (Fabre, Goulet et al. 2002). This process involves the transfer of LTA4 from a donor cell (typically a leukocyte such as a neutrophil) to a recipient cell (typically a non-leukocyte such as an endothelial cell), which can convert it to, for example, LTC4 (Okunishi and Peters-Golden 2011).

The leukotriene biosynthesis cascade begins with the synthesis of Leukotriene A4

(LTA4) by oxidation of arachidonic acid (AA) by 5-Lipoxygenase through a two-step reaction

(Rådmark, Werz et al. 2007). In the first step a hydrogen atom is abstracted from the C7 of

AA, followed by the addition of molecular oxygen, to generate 5-HpETE (5- hydroperoxyeicosatetraenoic acid). The second step involves removal of a hydrogen atom from

C-10, resulting in formation of the allylic epoxide LTA4 followed by the release of the product from the enzyme. LTA4 is a substrate for both LTA4-H (LTA4 hydrolase) and LTC4-S

[LTC4 (leukotriene C4) synthase]. LTA4-H generates LTB4 [leukotriene B4, 5(S), 12(R)- dihydroxy-6, 8, 10, 14-(Z, E, E, Z)-eicosatetraenoic acid] by the stereo specific addition of water to C-12. On the other hand, LTC4-S forms the product LTC4 [5(S)-hydroxy-6(R)-S- glutathyionyl-7, 9, 11, 14(E, E, Z, Z)-eicosatetraenoic acid], by addition of a glutathione at C-6

(Murphy and Gijon 2007). LTC4 is exported out of the cell where it can be processed by γ-

8 glutamyl transpeptidase to LTD4 and finally to LTE4 by dipeptidase hydrolysis. These three

LTs are also known as cysteinyl LTs owing to the fact that they all have a cysteine moiety attached to the C-6 via thiol linkage. (Figure 4).

Figure 4. Leukotriene Synthesis Cascade. AA is converted to LTA4 by the action of 5-LOX.

This is then converted to LTB4 by the enzyme LTA4 Hydrolase. LTA4 is also a precursor to cysteinyl Leukotriene (cysLT), LTC4 through addition of glutathione by the enzyme LTC4 Synthase. LTC4 can be further metabolized into other cystienyl LTs, LTD4 (by γ-glutamyl transpeptidase) and subsequently into LTE4 (by Dipeptidase).

LTs are extensive studied mediators of inflammation (Haeggstrom and Funk 2011).

LTs act by binding to specific G protein-coupled receptors that are located on the outer plasma membrane of structural and inflammatory cells (Peters-Golden and Henderson 2007).

The different LTs can have distinct cellular targets and activities such as smooth muscle cell contraction by cysLTs and neutrophil chemotaxis (Palmblad, Malmsten et al. 1981) by LTB4.

Specifically, LTB4 is a potent mediator of innate immune response (Soares, Mason et al.

2013). It can induce intracellular signaling cascades leading to activation of inflammatory cells by binding to cell surface receptors BLT1 and BLT2 (Hicks, Monkarsh et al. 2007,

Back, Powell et al. 2014). LTB4 also induces cell aggregation, increases vascular permeability (Smith 1981) and aids the adherence of neutrophils to the vascular walls

(Afonso, Janka-Junttila et al. 2012).

The cysLTs are involved in anaphylaxis and are important effectors of allergic diseases.

They may play a role in pulmonary distress due to powerful broncho-constrictor properties

(Dahlen, Hedqvist et al. 1980), ability to cause bronchial edema (Dahlen, Bjork et al. 1981) and increasing mucus secretions (Peatfield, Piper et al. 1982, Kaliner, Shelhamer et al. 1986).

They have also been implicated in bronchial asthma (Singh, Tandon et al. 2013), rhinitis

(Cobanoglu, Toskala et al. 2013) and allergic eye disease (Gane and Buckley 2013).

1.4 Human 5- Lipoxygenase - relationship between structure and function:

The human 5-lipoxygenase is a 673 aa, Ca2+ activated (Hammarberg, Provost et al.

2000), membrane binding enzyme, which is aided by the proteins FLAP (5-LOX Activation

Protein) (Abramovitz, Wong et al. 1993) and CLP (coactosin like protein) (Rakonjac, Fischer et al. 2006). However, the wild type 5-LOX posed considerable challenge for successful crystallization. To alleviate this issue a ‘Stable 5-LOX’ was engineered by removal of destabilizing effects of candidate membrane and Ca2+ binding amino acids via mutations

(Gilbert, Bartlett et al. 2011).

At first glance the structure of human 5-LOX exhibits all classical structural features seen in mammalian LOXs. The residues 1-112 form the primarily beta sheeted N-terminal

10 regulatory domain(Allard and Brock 2005) and the residues 125-673 form the much larger C- terminal catalytic domain. As expected, this domain houses the catalytic site with a catalytic iron forming the center of the catalytic action. The active site cavity appears to somewhat follow the current LOX structural consensus (Newcomer and Brash 2015) by having a partial

U shaped cavity lined with highly conserved Leu and Ile residues.

However, on close inspection, this structure presents a unique catalytic cavity. The catalytic center appears to be completely shielded from outer solvent state with no obvious substrate access site. The helix α2 is much shorter compared to what is seen in others with the side chains of residues Phe 177 and Tyr 181 positioned to impede access into the catalytic cavity (Figure 5). Exploring the possible role of this so called F-Y plug in substrate access and enzyme activity has been a major focus of my research presented below. The putative actions of the plug in overall enzyme activity have been referred as ‘corking’ and ‘decorking’ henceforth.

The catalytic Fe in this enzyme is held into place by coordination with side chains of three conserved His residues (numbered 367, 372 and 550) and the main chain carboxyl group of the C-terminal Ile 673(Gilbert, Bartlett et al. 2011). It may be also aided in this by the residue

Asn 554 (Figure 6), which is not close enough to be present in the actual coordination sphere. As far as the reaction mechanism is concerned this catalytic Fe acts as both an electron donor and acceptor during the course of the reaction (Figure 7).

Figure 5. Comparison of α2 helix of 5-LOX with other LOXs. (A) The helix in 5-LOX (blue) is much shorter as compared to the corresponding helices in 12-LOX (yellow) and 15-LOX (green). The ‘F-Y plug’ is shown in context to the catalytic Fe. (B) The position of the FY plug amino acid side chains (purple sticks) and their region of influence (purple mesh) is shown with respect to 5-LOX active site contour (blue).

Figure 6. Coordination of Catalytic Fe in 5-LOX. The Fe moiety (rust) is held in place by coordination bond formation with amino acid residues (green stick representation) in the catalytic center of 5-LOX. 12

Figure 7. The various states of catalytic Fe during 5-LOX action. The inactive enzyme (red boxed form) is activated (blue boxed form) by the lipid hydroperoxide (LOOH). The reduction state of Fe changes several times during the reaction ( indicated by the green arrows)[Adapted from (Murphy and Gijon 2007)].

As mentioned earlier, abnormal activity of 5-LOX has been implicated in in several disorders related to allergy and inflammation, such as asthma, rheumatoid arthritis, psoriasis etc.

(Henderson 1994). Products of 5-LOX pathway have also been associated with carcinogenesis

(Sethi, Shanmugam et al. 2012) and atherosclerosis in Humans (Jawien and Korbut 2010).

Needless to say these pathological effects of 5-LOX make it an attractive candidate for drug targeting. So far the only drug specifically targeting 5-LOX that has made to commercial distribution is Zileuton (Carter, Young et al. 1991). This leaves the field wide open for the development of more selective and effective drugs against 5-LOX.

13 As seen from the available literature, 5-LOX has far reaching effects in the coordination of physiological processes in a living individual. The various effects of 5-LOX activity in the

Human system makes it a promising drug target. Even though the structure of a stabilized form of this enzyme is available (Gilbert, Bartlett et al. 2011), the distinctive active site cavity and its apparent lack of an obvious entry point for the substrate gives us an incomplete picture. My research is aimed at enhancing our understanding of how this particular enzyme works and how it activity is controlled. This will not only help to further our knowledge about this important enzyme but will also aid in design and development of suitable pharmacological targeting candidates

14 CHAPTER 2. IDENTIFICATION OF THE SUBSTRATE ACCESS PORTAL OF 5-LIPOXYGENASE

2.1 Overview

The overproduction of inflammatory lipid mediators derived from arachidonic acid contributes to asthma and cardiovascular diseases, among other pathologies. Consequently, the enzyme that initiates the synthesis of pro-inflammatory leukotrienes, 5-lipoxygenase, is a target for drug design. The crystal structure of 5-LOX revealed a fully encapsulated active site and how the substrate gains access is unknown (Figure 8).

Figure 8. Possible substrate entry points in 5-LOX. The blue contour rendering of the 5-LOX active site indicates a completely enclosed active site. The possible sites for the entry of AA are indicated by green arrows and the likely amino acid side chains (purple) that might be involved in those sites are also indicated. Removal of the plug requires the movement of both main chains and side chains of the amino acids while for ‘portal’ entry only rotamer shift of the Trp 147 side chain is required.

We then asked whether a structural motif, aromatic “corking” amino acids, present in

5-LOX but absent in other mammalian lipoxygenases, might reposition to allow substrate access. Our results indicate that reduction of cork volume facilitates access to the active site.

However, if the site is obstructed, enzyme activity is severely compromised. Our results support a model in which the “corking” amino acids shield the active site in the absence of substrate, and help position the substrate for catalysis.

2.1 Introduction

The enzyme 5-lipoxygenase (5-LOX) initiates the biosynthesis of the eicosanoid lipid mediators known as leukotrienes (LT). Together with 5-Lipoxygenase-Activating Protein

(FLAP), localized to the nuclear membrane, 5-LOX transforms arachidonic acid (AA) to leukotriene (LT) A4 in a two-step reaction that proceeds via a hydroperoxy intermediate

(Haeggstrom and Funk 2011, Radmark, Werz et al. 2014). Downstream metabolites of LTA4 are potent signaling molecules involved in diverse processes, some of which are mediated by

G-Protein coupled receptors. For example, LTD4 and LTC4 induce bronchoconstriction upon binding to their cognate G-Protein Coupled Receptors in smooth muscle cells, while LTB4 is a potent leukocyte chemoattractant (Funk 2001). Due to its role in the production of inflammatory lipid mediators, 5-LOX is a target for the development of therapeutics for conditions as diverse as asthma, cardiovascular disease, (Pergola and Werz 2010, Steinhilber and Hofmann 2014) pancreatic cancer (Sarveswaran, Chakraborty et al. 2015) and traumatic brain injury (Corser-Jensen, Goodell et al. 2014).

Lipoxygenases are non-heme iron dioxygenases that catalyze the peroxidation of polyunsaturated fatty acids (Brash 1999, Kuhn and Thiele 1999). Humans express six

16 lipoxygenases (Funk, Chen et al. 2002), and each is named according to the site of peroxidation of AA. Thus, 5-LOX produces the intermediate 5-hydroperoxyeicosatetraenoic acids (5-HpETE), whereas, for example, a 15-LOX produces 15-HpETE. Thus while animal

LOXs metabolize a common substrate, they differ in product specificities.

The product generated is both stereo-specific and regio-specific, as either a specific

R- or S- isomer is produced even though the substrate of choice AA has 3 possible sites for hydrogen abstraction and thus can theoretically generate 12 possible products (Figure 9).

Figure 9. Arachidonic Acid as a substrate. The red blue and green colored regions indicate the three chemically equivalent carbons and their associated hydrogens available for abstraction. The possible products with their sterio and region identities are color coordinated

Both the 5- and 15-enzymes produce the S-isomers of their respective products. The stereo-chemistries of these two products are consistent with two distinct modes of substrate binding (Schneider, Pratt et al. 2007) (Figure 10) corresponding to head-first (carboxyl) or tail-first (hydrocarbon) entry into the active site (Egmond, Vliegenthart et al. 1972, Coffa,

Imber et al. 2005).

One of three AA pentadienes must be positioned with its methylene carbon accessible to the catalytic machinery for H abstraction, and H abstraction generates a free radical that is accessed by O2 from the opposite face of the AA. The stereo-chemistry of the product is a consequence of this antarafacial relationship between the catalytic iron and the O2 access channel/pocket.

Figure 10. LOX product stereo-chemistries are consistent with one of two orientations of AA is the active site. (a) Contour of the active site (shaded region) of 15-LOX-2 (pink, 4NRE) as defined by a competitive inhibitor in green. The α2 helix of 5-LOX in blue cartoon rendering and stick rendering of the F-Y plug is superimposed on the 15-LOX-2. (b) The equivalent rendering of 5-LOX (blue, 3O8Y). (c) For cavities of equal depth, inverse positioning of the substrate generates the 15-S- “tail-first” product or the 5-S “head-first” product.

Several LOX structures are available and combine to provide details of a consensus active site (Newcomer and Brash 2015), but the 5-LOX isoform differs significantly from 15-

LOX-1 (Gillmor, Villasenor et al. 1997, Choi, Chon et al. 2008), 15-LOX-2 (Kobe, Neau et al.

2014), 12-LOX (Xu, Mueser et al. 2012) and 8R-LOX (Oldham, Brash et al. 2005, Neau,

Bender et al. 2014) in three important ways: (1) The HPETE produced by 5-LOX is an intermediate; a second reaction in the same 5-LOX active site converts the intermediate to

LTA4. (However, it should be noted that in vitro 15-LOX-1 can also utilize 15-HPETE as a substrate and generate a product analogous to LTA4 (Bryant, Schewe et al. 1985) , (2) Only 5-

LOX requires accessory proteins for maximal activity: an AA -binding protein(FLAP) embedded in the nuclear membrane (Dixon, Diehl et al. 1990, Evans, Ferguson et al. 2008) and a cytosolic coactosin-like protein (Rakonjac, Fischer et al. 2006, Basavarajappa, Wan et al.

2014) and (3) 5-LOX displays a distinctive structural variation on the canonical LOX fold.

In general the relative placements of the roughly 20 α-helices that make up the catalytic domain are conserved among LOX isoforms. 5-LOX, as well as an 11R-LOX from

Baltic coral (Eek, Jarving et al. 2012), displays alternate placement of helix α2. This helix rims the U-shaped active site in other isoforms, but in 5-LOX (and 11R-LOX) the helix is “broken” to seal it off with insertion of aromatic amino acids into an otherwise fully accessible U-shaped active site. Thus the structure of Stable-5-LOX (so-called because of the presence of stabilizing mutations introduced distal to the active site) revealed a fully encapsulated catalytic machinery and neither entry into, nor positioning of substrate in the active site are obvious in the reported structure (Gilbert, Bartlett et al. 2011).

We used site-directed mutagenesis, coupled with substrate and product analyses using

High Performance Liquid Chromaography (HPLC) (as depicted in Figure 11), to sample

19 possible entry portals that may permit the substrate to gain access to the sheltered catalytic machinery and position itself in the active site. We also monitored the structural and functional integrity of the mutants generated by Thermal Shift Assay (TSA) (Lavinder, Hari et al. 2009) and Enzyme Kinetics (EK) measurements

Figure 11. Schematic of HPLC analysis and sample chromatograms. HETE product profiles from incubations of Y181A, F177A and F1771A:Y181A mutants are shown.

The different possible regions of substrate entry also put in to question the actual orientation of the substrate entry so as to generate the product of correct region- and stereospecificity. In other words, whether the substrate is entering carboxyl end or the methyl end first also dependent on which site of the active site is the chosen entry point. To test this we used several substrate analogs of AA with varying levels of bulk at the carboxyl end 20

(figure12). The principle behind this approach was that if the substrate approaches via the carboxyl end then a bulkier group will prevent the formation of the product. Also utilization of such bulkier substrate will indicate a larger opening of the point of entry at the active site.

Figure 12. Comparison of AA and substrate analogs. The natural substrate AA along with the several substrate analogs used for the study. The differences from AA are highlighted in red

Our results support a model in which active site access appears to require repositioning, rather than a complete displacement of, Phe-177 and Tyr-181, which penetrate the active site cavity. Although paring down the “cork” volume may facilitate entry into the active site, obstruction of “cork” entry into the active site leads to loss of enzyme specificity, stability, and activity.

21 2.3 Results

We sampled, via site-directed mutagenesis, amino acids that line the enclosed active site cavity of 5-LOX (Figure 13) to determine what role, if any, they might play in allowing

AA to gain access to the catalytic iron and position itself in the active site such that 5-HpETE and LTA4 can be produced.

Figure 13. Amino Acids in Stable-5-LOX targeted for Site-Directed Mutagenesis. The active site contours a rendered in blue and the amino acid side chains are shown in light blue stick rendering

Mutations were made to Phe-177, Tyr-181, Ala-603, and Trp-147. The first three cluster to truncate one arm of what has been described as a U-shaped cavity in homologous enzymes with accessible catalytic sites, 15-LOX types -1 (Choi, Chon et al. 2008) and -2

(Kobe, Neau et al. 2014), 12-LOX (Xu, Mueser et al. 2012) and 8R-LOX (Oldham, Brash et al.

2005, Neau, Bender et al. 2014). Phe-177 and Tyr-181 form what has been described as the active site “cork” (Gilbert, Bartlett et al. 2011) and the small size of the Ala at 603 appears to allow insertion of the “cork” into the active site. In contrast, Trp-147 appears to block cavity accessibility via a sub-cavity at the base of the U (Figure 14).

Figure 14. Stable-5-lipoxygenase has an encapsulated active site. (a) Cartoon rendering of 5- lipoxygenase (3O8Y). There iron is shown as an orange sphere. The amino acids mutated are in sick rendering (C, pink, O red, N blue). (b) Close-up of the internal cavity with the mutated amino acids in stick rendering.

Each of the mutants was evaluated in terms of kinetic parameters and product profiles.

While the in vivo product of the 5-LOX reaction is LTA4, the capacity of 5-LOX to convert the

5-HPETE intermediate to the leukotriene is significantly compromised in vitro due to the

23 absence of FLAP. Thus, kinetic parameters derived from initial reaction rates are reported for

HPETE formation, monitored at 238 nm as depicted in Figure 15 [The hydroperoxy products are rapidly reduced to the corresponding hydroxyeicosatetraenoic acids (HETEs). Products are expressed as ratios relative to the Prostaglandin B1 (PGB1) standard. For simplification, the products will henceforth be referred to as HETEs].

Figure 15. Measurements of Enzyme Kinetics. (A) Schematic showing the measurement procedure (B) Sample Velocity vs Substrate concentration plots of Stable 5-LOX compare to Y181A mutant to indicate difference.

Mutations to the “plug” cluster had dramatic effects on enzyme activity. The plug was trimmed by removal of either or both of the aromatic side chains (Y181A or F177A) (Figure 16). This increased 5-HETE yields from enzyme-substrate incubations ~3- and ~1.5-fold, single and double mutants respectively. However, this detected increased yield came at a cost 24 to product specificity, as a small fraction of HETE other than 5-HETE was produced by Y181A (figure 17). The stable 5-LOX and the wild type 5-LOX do not produce any other HETEs under normal circumstances.

Figure 16. A structural schematic of effects of trimming the ‘plug’ (rendered using PyMOL program) (A) Mutant F177A, figure scheme same as Fig. 5. Mutant amino acid is in green stick rendering; (B) Mutants Y181A; (C) Mutant F177AY181A, mutant amino acid shown in light teal.

Figure 17. Effect of trimming the ‘plug’ on the 5-LOX activity. HETE production by different plug trimming mutants relative to PGB1 levels

Neither the Y181A nor the F177A mutation appears to have an impact on enzyme stability, since thermal denaturation measurements revealed no significant change in Tm

(Figure 18). Kinetic analyses indicate that, while the Km remains essentially unchanged, the turnover rate for Y181A is roughly 50% enhanced with respect to Stable-5-LOX. Thus

25 “removal” of the bulky Tyr is consistent with an enhanced entry and/or egress from the active site. The F177A and Y181A double mutant resembles the F177A single mutant with respect to reaction rate as well as substrate and product specificities. Notably, though, the double mutant has greater thermal stability than either of the single mutant forms (Figure 18).

Figure 18. Effect of trimming the ‘plug’ on the 5-LOX structural and functional integrity (A) Shift in Tm of the mutants as compared to stable 5-LOX, indicative of structural integrity. (B) Enzyme kinetic measurements comparing Km and kcat values of Stable 5-LOX with the mutants indicating changes in functional integrity.

LTA4 as well as di-HETEs that may form from 5-LOX recognition of HETEs as substrates have absorption maxima at 270-280 nm and UV spectra that clearly differentiate them from HETEs. The time frame of the kinetic measurements minimizes signal interference from di-HETE formation, but these off-pathway products do accumulate in the course of the incubations. LTA4 breakdown products and di-HETEs, both a consequence of 5-LOX activity with a product HETE, are grouped together as “other” in the product analyses in Table 2 and

Figure 19.

The presence of F177 appears to be necessary for utilization of 5-HETE as a substrate, as none of the mutants in which F177 was replaced with Ala produced compounds that might

26 be leukotrienes or di-HETEs, despite the fact that the F177A mutant generates 50% more 5-

HETE than its progenitor (Figure 18)

Y181A and F177A mutants were able to utilize AA analogs with bulky groups at the carboxyl end, arachidonyl-ethanolamine (AEA) and N-arachidonyl-glycine (NAGly), to some extent (Table 2)

Figure 19. Utilization of 5-HETE as a substrate by ‘plug’ mutants. Levels indicated as ratio of products detected at 270nm UV to PGB1.

Table 2 Utilization of substrate analogs by ‘trimmed’ mutants

Mutant St-5-LOX Y181A F177A F177A Y181A

DGLA X X X X AEA X X   NAGly X   

We then asked whether impeding “plug” insertion by mutation of A603 might have a similar impact to paring down the “plug.” A large amino acid side chain at this position, as is found in the homologous LOXs with open cavities, should preclude cork insertion because of its proximity to Tyr-181 (Figure 20).

Figure 20. Effect of unplugging the ‘plug’ on 5-LOX activity. HETE production by different plug trimming mutants relative to PGB1 levels.

Table 3. Utilization of substrate by ‘unplugged’ mutants

Mutant St-5-LOX A603L A603L Y181A

DGLA X  

AEA X  

NAGly X  

In contrast to the mutations that minimize cork volume, the A603L mutation resulted in

◦ significant destabilization of the protein with a 4 C drop in the Tm. While the Km for AA is native-like, the enzyme turnover is less than 10% of the parent enzyme. Moreover, the enzyme has significantly impaired substrate specificity and is able to oxygenate arachidonic acid

28 analogs that are not substrates for the wild- type enzyme (Figure 20). Table 3 lists the behavior of the mutants with substrate analogs.

Figure 21. Effect of unplugging the plug on the 5-LOX structural and functional integrity (A) Shift in Tm of the mutants as compared to stable 5-LOX,(B) Enzyme kinetic measurements.

Remarkably, we can recover a native-like kcat (0.7 vs. 0.8 s-1) by making a compensatory mutation: The substitution of Y181 with Ala to whittle down the cork and allow insertion of Phe-177 despite the added bulk at position 603. This double mutant

(Y181A/A603L) has enhanced thermal stability, and shows a slight decrease in Km (~2 fold) relative to Stable-5-LOX (Figure 21). HPLC traces of the different HETEs formed by the different A604 plug mutants are shown in figure 19 compared to HETE standard trace. As can be seen from the traces that, although the A604LY181A double mutant makes high amounts of

5-HETE, it also makes some amounts other different HETEs like 15-, 12/8- and even 11- , which is not generally seen as a product in animal LOXs.

Figure 22. HPLC traces of unplugged mutants. Relative HETE formation by Stable 5-LOX mutants A604L (Orange) and Y181AA604L (peach) compared to HETE standards (grey)

While opening the “corked” portal seemed the more likely entrance because the open conformation would conform to what has been observed in other LOX structures, we also tested a possible access portal obstructed by W147. The substitution of Trp with the smaller

Leu led to a significant reduction in enzyme stability.

In addition, the enzyme has significantly reduced activity and the product specificity is compromised (Figure 23). These characteristics are inconsistent with Trp-147 serving as a functional portal. The entire data as obtained by the different ‘plug’ mutants is summarized in table 4 a. & b.

Figure 23. W147L mutant destabilizes the enzyme both structurally and functionally. (A) Shift in Tm of the mutants as compared to stable 5-LOX, (B) Enzyme kinetic measurements

Table 4a. Summary of Thermal Shift and Enzyme Kinetics measurements of ‘plug’ mutants.

Mutant St-5-LOX Y181A F177A F177A A603L A603L W147L Y181A Y181A

Tm (°C) 53.1±0.6 52.9± 0.1 51.7±0.1 54.8 ±0.5 49.1±0.3 55.8 ±0.6 49.9 ±0.1

Kinetic parameters

Km(µM) 14±1.6 7.6± 3.4 9.8±2.1 11±2 9.2±3.2 5.0±2.8 22±7 kcat (s-1) 0.8±0.03 1.2± 0.2 0.7±0.04 0.7±0.03 0.05±0.01 0.7±0.1 0.01±0.01

Table 4b. Summary of product formation by ‘plug’ mutants.

Products

5-HETE 4.1±0.2 11.1±0.3 5.8±0.3 3.1±0.4 0.13±0.1 7.7±0.03 1.7±0.6

11-HETE nd nd nd nd nd 0.57±0.02 nd 8/12- HETE nd 0.27±.04 nd nd 0.22±.04 1.9±.06 0.6±.05 15-HETE nd nd nd nd nd 0.66±.05 0.17±.01 “other” 0.6±0.04 0.4±0.03 nd nd nd 4.1±0.5 0.25±0.1 nd- Not detected

A second question to address was whether AA is positioned with its carboxyl innermost, as has been inferred from the S-stereochemistry of the product. Extrapolating from the structure of 8R-LOX in complex with AA (Neau, Bender et al. 2014), His-600 is deep in the active site where the tubular cavity dead-ends (Figure 24).

Figure 24. Position of His600 in Enzyme Active Site. (A) The dotted arrow indicates the possible orientation of AA in the active site with the arrowhead signifying the putative position of the carboxyl group. (B) Position of His 600 (green sticks) with respect to the F-Y plug (blue sticks).

We tested whether substitution of this charged amino acid might impact 5-LOX activity with two mutations: H600A and H600V. Multiple substitutions of non-polar amino acids are important at this position, because the depth of the active site cavity determines whether the pentadiene to be attacked can be positioned productively at the catalytic iron. Both substitutions resulted in proteins which were expressed and purified at levels comparable to that of the parent enzyme, and had Tms that exceed that of the parent protein. H600A displays severely compromised enzyme activity, in terms of both Km and turnover (Figure 25).

Figure 25. Effect of different His600 mutants on Thermal Shift and Enzyme Kinetic measurements A) Shift in Tm of the mutants as compared to stable 5-LOX, (B) Enzyme kinetic measurements as compared to stable 5-LOX. His600V had no detectable enzyme activity.

The low level of activity of the mutant precluded product profile analysis by HPLC.

The H600V mutation resulted in total lack of catalytic activity. However, we were able to rule out that the catalytic center is non-functional in the mutant with a “rescue” mutation that combined H600V with F177A:Y181A. This triple mutant produced HETEs, but not the 5- or

15- HETE. Instead products consistent with attack at C10 were observed. Thus H600 appears to be essential to proper positioning of the substrate (Figure 26). The results are also summarized in Table 5.

Figure 26. Product formation by the triple mutant as compared to stable 5-LOX. The triple mutant only makes 12/8 HETE as opposed to 5 HETE.

Table 5. Summary of the different properties of His600 mutants

Mutant St-5-LOX H600V H600A F177A:Y181A:H 600V

Tm (°C) 53.1±0.1 56.4± 56.7± 54.8 (0.1) 0.2 0.2 Kinetic parameters

Km (µM) 14±1.6 nd 27±0.7 5.4±0.5 Kcat (s-1) 0.8±0.03 nd <0.01 0.3±0.01 Products 5-HETE 4.1±0.2 nd nd nd 8/12- HETE nd nd nd 3.4±.02 15 HETE nd nd nd nd “Other” 0.6±0.04 nd nd nd

The ability of a LOX to metabolize an AA analog which carries a bulky group at the carboxyl has been used as a proxy for “tail-first” entry as “head-first” entry would require that the additional bulk be accommodated in the active site, but with tail first entry the extra cargo need not enter the active site. 5-LOX does not recognize AEA or NAGly as substrates, consistent with head-first entry. However, several of our mutants are able to recognize these substrates. How might that be possible? Note in Fig 9a, 5-LOX A603 is positioned between the top of the U-shaped cavity of 15-LOX-2, between the two arms, with Tyr-181 simultaneously truncating the outermost arm and closing off the innermost arm. In 5-LOX (Fig. 9b), the deeper end is curved toward the entrance end such that removal of Tyr-181 might allow the ends of the U to join to form an egg-shaped cavity.

Thus it is likely that Y181A mutants tolerate extra bulk on the carboxyl end of a substrate analog because of the volume vacated by the Tyr. Thus, the ability to metabolize

AEA or NaGly is not indicative of the methyl end of the substrate situated innermost, but simply a consequence of a roomier cavity that allows “head-first” entry with added bulk.

2.4 Discussion

Lipoxygenases are characterized by two domains (Boyington, Gaffney et al. 1993,

Gillmor, Villasenor et al. 1997, Newcomer and Brash 2015): a β-barrel domain which is implicated in membrane binding and a much larger α-helical domain that harbors the catalytic iron. Invariant histidines, as well as the main chain carboxyl of the terminal invariant Ile position the iron. 5-LOX displays a distinct variation of the typical LOX fold. While the structures of 15-LOX-2, 15-LOX-1, 8R-LOX, and 12-LOX have revealed a solvent accessible active center at the base of a U-shaped cavity, access is firmly “corked” in 5-LOX by the insertion of the side chains of Phe-177 and Tyr-181. Thus the path of substrate entry is not apparent in the crystal structure. We asked whether 5-LOX may undergo a conformational change to “uncork” the active site by site-directed mutations that (1) reduce of cork volume and/or (2) hinder “cork” insertion.

Active site access

Our data are most consistent with a model in which the cork must reposition, but it is not fully ejected from the binding site. The cork is comprised of two aromatic amino acids

(F177 and Y181), and while our data indicate that replacement of either or both with Ala results in catalytically active mutants, Y181A has significantly increased activity. This suggests that Y181 is dispensable for the catalytic reaction, but in the native enzyme it may

35 limit access to the active site. The increase in activity, reflected by a 50% increase in kcat, is consistent with facilitated entry/egress to/from the active site.

An increase in the rate of HPETE production by an R101D 5-LOX mutant was recently reported. This mutation disrupts a salt-link between the tightly associated membrane binding domain and the catalytic domain which is mediated by Asp-166 (Rakonjac Ryge, Tanabe et al.

2014). The proximity of the corking amino acids to this domain interface is consistent with this observation, in the sense that disruption of the interface could allow greater flexibility in the corking peptide.

While our data support repositioning of the “cork.” it also indicates that the entire cork cannot be displaced, as a mutation that blocks cork insertion displays less than 10% of the activity observed for the parent enzyme. The mutation A603L, designed to interfere with cork insertion due to the proximity of Tyr-181 to Ala-603, leads to a nearly total loss of activity, indicating that a cork amino acid side chain is necessary during the catalytic cycle. A “rescue” mutation, in which substitution of Y181 of the cork with Ala relieves the steric clash with the added bulk at position 603, renders a highly active enzyme, albeit with compromised product specificity. In addition, the cork amino acid F177 appears to be required for 5-LOX to process

5-HETE, which it must do to complete the transformation of AA to LTA4. No metabolites of 5-

HETE are observed in any of the mutant forms that lack Phe-177.

Previous mutagenesis studies aimed at reducing the volume of the active site of mouse and human 5-LOX have explored single site mutants at Ala-603 (Schwarz, Gerth et al. 2000,

Schwarz, Walther et al. 2001, Hofheinz, Kakularam et al. 2013). The investigators observed that substitution with Ile was tolerated at this position, however for the human enzyme a

36 reduction of product isolated from cell lysates (relative to that isolated for the bacterially expressed wild- type enzyme) was observed. In addition, loss of regio-specificity was reported.

In contrast, substitution with Phe at this position led to a total loss of enzyme activity. These results can also be interpreted the structural context we propose: that addition of bulk at position 603 interferes with the insertion of Phe-177/Tyr-181 in the active site and can lead to significantly compromised enzyme activity.

Head first vs. tail first entry

Further experiments suggest that His 600 is essential for positioning of the substrate, as mutations at this position abolish enzyme activity. The amino acid is deepest is an elongated cavity that must accommodate the 20 carbon substrate. A wealth of biochemical data are consistent with AA entering “head” (carboxyl) first into the 5-LOX active site, while in the homologous 15-LOX-2 (42% sequence identity), the “tail” (hydrocarbon) enters first. This

“head-first” vs “tail-first” relationship is a crucial determinant of product stereo-chemistry as it places the carbon for peroxidation proximal to the O2 channel (Schneider, Pratt et al. 2007,

Newcomer and Brash 2015).

One can see from Fig.10 that the 5-S and 15-S products have their peroxide groups on opposite sides of the product, consistent with the inverse relationship of AA positioning in the active site. We observed that mutation of His-600 to either a Val or an Ala resulted in an enzyme that can no longer generates 5-HPETE, presumably because the absence of a neutralizing amino acid deep in the cavity results in an enzyme that no longer favors “head- first” substrate entry.

However, in a similar experiment with mouse 8-S-LOX, the substitution of the His at the deep end of the pocket resulted in the generation of a product consistent with tail-first entry

(Jisaka, Kim et al. 2000). So why is a “tail-first” product, which should be a 15-HETE according to Fig 10, not observed for the 5-LOX H600A or H600V mutations? Cavity depth is an essential aspect of LOX structure as it allows the substrate to slide into the active site and position the pentadiene for attack. Cavity depth is clearly impacted when His is replaced by

Ala or a Val, and one possibility that no product is observed because there is simply no productive alignment of a pentadiene at the catalytic center.

Thus we combined the His600V with mutations that permit slack in the AA binding site by opening the “corked” end. F177A:Y181A “rescues” the H600V mutation, recovering ~50% of the parent enzyme activity. However, no 5-HETE is produced. Only products consistent with attack at carbon 10 are isolated from the incubations. Thus H600 does not play a catalytic role, but it is required for productive alignment of the substrate.

Why is the active site of 5-LOX “corked”?

Recent structures of lipoxygenases have combined to provide a robust model for understanding product specificity in this family(Newcomer and Brash 2015) and suggest a U- shaped cavity with a highly conserved core of amino acids that position the appropriate pentadiene for attack.

It is not entirely clear from the consensus model is why the active site of 5-LOX is

“corked” since Phe-177 is a highly conserved amino acid whose counterparts in other arachidonate metabolizing LOX do not block access to the U-shaped channel. Instead they abut the active site cavity with the plane of the aromatic ring perpendicular to the fatty acid tail

(Figure 27). The structures of 15-LOX-2 and 5-LOX (~42% sequence identity) superimpose with an r.m.s. of 1.17 Å for 576 of 674 Cα’s, but the distance between the Cα’s of Phe-177 and its equivalent in 5-LOX-2 (Phe-184) is ~8 Å.

Figure 27. Displacement of the corking residues in 5-LOX. 5-LOX “corking” amino acids are displaced relative to their conserved counterparts F175, L179 (gold, lavender) in 12-LOX (3RDE) and rabbit 15-LOX (2P0M), respectively; F184,A188 in 15-LOX-2 (green, 4NRE) and Y178,R182 in 8R-LOX (white). AA (white) and 12-LOX (orange) and 15-LOX-2 (pink) inhibitors are rendering in sticks.

This major shift in side chain placement is a consequence of an unraveling of α2 in 5-

LOX so that the helical segment that contains Phe-177 and Tyr-181 is displaced in this isoform and these side chains can insert into the active site. Notably, substrate or inhibitors shield at least 50% of the accessible surface area of the Phe-177 counterparts in 8R-LOX, 12-LOX or

15-LOX-2, i.e they form part of the wall of the active site and even in their “open” conformations play critical roles in substrate recognition. In contrast the counterparts of Tyr-

181 are largely solvent accessible amino acids at the entrance to the site. However, the 8R-

LOX counterpart of Tyr-181 (Arg 182) contributes significantly to positioning of substrate in a productive conformation (Neau, Bender et al. 2014), presumably because it provides a counter charge to the substrate carboxylate. However, a charged amino acid is not conserved in other tail-first LOX’s with open cavity access. Thus the amino acids at the periphery of the binding site can vary in placement and play distinct roles in binding site definition.

In conclusion, our results are consistent with a model in which the substrate gains access to the 5-LOX active site via a “cork,” which must be repositioned to allow substrate access. Phe-177 is likely to form an integral part of the active site, and His-600 is required to position the substrate for 5-HETE production.

2.5 Materials and Methods

Plasmid preparation

The Stable-5-LOX construct in a pET-14b (Novagen) plasmid was used as the starting point for mutation (Gilbert, Bartlett et al. 2011). The various mutations were introduced by site-directed mutagenesis. Primers were designed to contain a given mutation and then used to amplify the 5-LOX plasmid using whole plasmid PCR. The resultant products were gel purified and re-circularized. The primers also contained unique restriction sites as silent mutations to verify mutation. The presence of mutations was confirmed by DNA sequencing.

Protein expression and Purification

E.coli Rosetta2 (Novagen) cells were transformed with the mutated plasmids and grown in small scale cultures to check for protein expression and solubility via auto-induction

(Studier 2005). For large scale protein purification the cells were grown in Terrific Broth

(Alpha Bioscience, MD) with 25μg/ml chloramphenicol, 100μg/ml ampicillin and 5mM

MgSO4. The cultures were shaken at 250 rpm for 4 hours at 37°C and then the temperature was reduced to 25°C for an additional 25 hours to induce leaky expression. The cells were pelleted by centrifugation and frozen at -80°C. For protein purification, the cells were suspended in

3ml/gm Bugbuster (Novagen) with 1μM pepstatin, 1μM leupeptin, 100μM PMSF and 2KU/gm

DNAse I (all from Sigma). The mixture was stirred on ice for 30 min and lysed by passing through a French pressure cell. The lysate was centrifuged at 40000 g for 45 min.

The resulting supernatant was loaded onto a HisTrap 5ml cobalt Sepaharose (GE

Healthcare) column equilibrated in 50mM Tris (pH 8.0), 500mM NaCl, and 20mM imidazole on an AKTA FPLC system (GE Healthcare). Subsequently, the column was washed with ten column volumes of the above buffer and then the protein was eluted by applying a linear gradient of 50mM Tris-HCl (pH 8.0), 500mM NaCl and 200mM imidazole. The peak fractions were concentrated in an Amicon Ultra 30K (Millipore) filter to a final volume of 500 µl.

Buffer exchange was performed twice with 15ml of 20mM Tris-HCl, pH8.0, 150mM NaCl and

5mM TCEP-HCl. Protein was concentrated to a final concentration of 1mg/ml to 5mg/ml as detected by a NanoDrop spectrometer. The protein was flash frozen in liquid N2 and stored at -

80°C until usage.

Kinetics Assays

The kinetic parameters for Stable-5-LOX and the different mutants were determined by monitoring the increase in absorbance at 238 nm in an Agilent 8453 Diode Array

Spectrophotometer (Agilent Technologies, Santa Clara, CA, USA). The assays were performed with 500nM enzyme in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl and 5mM TCEP. Substrate concentration was varied from 5 to 100 μM. Substrate concentrations (5, 10, 20, 30, 40, 50,

100 µM) were monitored in triplicate for each sample. Km and Vmax values were determined by non-linear regression analysis of a plot of velocity versus substrate concentration to equation 1.

푉max is the maximal velocity, 푆 is the substrate concentration, 퐾m is the Michaelis constant.

푉max∙[푆] Equation 1 푣 = 퐾m+[푆]

Product Assays

The mutant enzymes (5μM) were incubated at 37°C with 100μM substrate or substrate analog (dihomo-γ-linolenic acid (DGLA), N-arachidonyl glycine (NaGly), anandamide (AEA), docosahexanoic acid (DHA) (all from Cayman Chemical Company) in 20mM Tris-HCl. (pH

7.5), 150mM NaCl, and 5mM TCEP-HCl to a final volume of 200μl for 5min. Prostaglandin

B1 (5μl of 50ng/μl, Cayman) was added after completion of the incubation as an internal standard. The products of the reaction were extracted by addition of 2μl 1N HCl and 20μl 1M

K2HPO4 followed by 400ul dichloromethane. The mixture was vigorously shaken for 10 sec and centrifuged briefly to separate the phases. The organic phase was collected and washed twice with 400μl water, dried under a stream of N2 and stored at -20°C.

The products were re-suspended in 30ul of 50% Methanol and reduced by addition of triphenylphosphine and loaded onto a Supelco Discovery C18 Column (5μm particle size, 25 x

0.46 cm, Sigma-Aldrich) for identification and quantitation with reversed phase HPLC with a diode array detector (Dionex). For quantitation, peak areas are expressed relative to that of the

PGB1 internal standard. The mobile phase was acetonitrile/water/formic acid (60:40:0.01), and the flow rate 1ml/min. Four UV channels (220, 235, 270 and 280 nm) were monitored.

Melting temperatures

Melting temperature assays were performed as describe by Ericsson et al (22). Briefly, samples were prepared with 2µM enzyme in 20 mM Tris-HCl (pH 7.5), 150 mM NaCl and

5mM TCEP and 1X SYPRO Orange stain (Invitrogen) in a final volume of 150 µL. Triplicate volumes of 40 µL each for every individual enzyme sample were aliquoted into a 96 well reaction plate. The plate was subjected to a temperature gradient of 5-94°C in 1° increments in a 7500 Fast Real-Time PCR system (Applied Biosystems).

The thermal denaturation of the proteins was monitored by fluorescence of SYPRO

Orange that occurs with protein binding (Figure28). The resulting data was exported to

SigmaPlot (v. 9) and averaged for each triplicate. The average sigmoidal part of each curve was then fit into a four parameter sigmoidal equation. The Tm value was generated using the above. The Tm values were measured in triplicate for each mutant. A mean of the Tm values with associated standard deviations (S. D) were obtained use Microsoft Excel software and have been reported for each mutant in Tables 4a and 5.

Figure 28. Thermal Shift Assay (TSA) procedure. (A) Schematic showing the TSA procedure in brief (B) Sample fluorescence intensity vs temperature traces of stable 5-LOX and H600A mutant showing a shift in Tm, possibly due to the mutation.

CHAPTER 3. CONCLUSIONS

The presence of several LOX isoforms in mammals in general and humans in particular is indicative of the important role this enzymes plays in smooth running of the physiological machinery. The widespread involvement of LOXs in various cellular functions only underscores the significance of this enzyme family in a living system. The spectrum of diseases associated with imbalance in the levels of LOXs in an organism makes them fitting drug targets. For successful drug designing it is imperative that every aspect of these enzymes are well known. A good starting point is looking at the structural and functional properties of the individual enzymes.

The enzyme five lipoxygenase has been extensively studied and linked unequivocally to chronic and acute inflammation. The enzymatic action and the stereo specificity of 5-LOX depends not only on the actual orientation of the substrate AA while entering the active site, but also the actual positioning of one of the three chemically equivalent pentadienes present with respect to the catalytic Fe. However, in the current available structure indicates a lack of an obvious site for the substrate AA to approach the active site. And even though the catalytic site is somewhat similar to what is commonly seen in LOXs, the unique shape of the catalytic cavity also poses a potential issue regarding the proper positioning of the substrate.

My research is an attempt to shed some light on these aspects by looking at the effect of site directed mutagenesis on candidate amino acid moieties in the active site. I looked at the effect of these single or combined amino acid mutations on both the substrate and the product specificity. My research indicates that an initial displacement of the so called ‘F-Y plug’ is required for the entry of the substrate. However for preservation of both substrate and product

45 specificities, this plug needs to be able to close back in. This is indicated by the alterations of both in 5-LOX mutants where the plug either rendered partially or completely unable to close.

I also attempted to look at the possible role of the His600 moiety positioned at the blunt end of the active site in the enzyme activity (Figure 29). Even though the two different amino acid mutants had proper folding and stability as inferred from the higher than usual Tms and yields comparable to their active counter parts, they had very little or low activity. The effect of this particular mutation was somewhat alleviated by combination with F177AY181A. The resulting enzyme showed activity as indicated by detectable HETE products in HPLC traces.

Nevertheless, 5-HETE was not produced by this mutant.

Figure 29. Position of His 600 at Enzyme Active Site. The position of His600 is shown here with respect to the active site and the catalytic Fe (red sphere). The residues Phe177 and Tyr 181 are also shown for give an idea of its overall placement.

These results suggest a possible role of this His in proper positioning of substrate. It seems that in the single His mutations the carboxyl group of AA cannot properly fit in the active site, thereby affecting the positioning of C7 for attack. The combination mutant, perhaps owing to ease of active site access, showed products that are indicative of attack on the C10.

This might suggest that the substrate is entering methyl end first instead.

The final piece of this puzzle would be the elucidation of how Oxygen is able to access the substrate. We tried to answer this question by mutating a His residue at position 432. As indicated from the active site structure this His close to the catalytic Fe in position and appears to be a possible gate for the oxygen channel. The product assays reveal that the mutants have some impairment in product specificity. The H432N had comparatively lower activity and produced more 12/8 HETE product than 5HETE. The H432A mutant mainly produced 5HETE with almost similar levels of 12/8 HETE production. This mutant also had higher activity. As seen by the results of the product assays on the Histidine mutants (Figure 27), the role of this particular His in oxygen channeling remains inconclusive and open to further research.

Figure 30. Product assays of His432 mutants. Relative HETE formation by Stable 5-LOX mutants H432A (Blue) and H432N (Red) compared to HETE standards(grey).

To conclude, my research has endeavored to explain the basis of specificity of the Human

5-LOX and it has elucidate certain key aspects of the structural control of specificity. The inconclusive nature of some mutants’ indicates that further research into the enzyme activity, especially oxygen channeling is essential. The most helpful of these would be a crystal structure of the enzyme bound to the substrate. This particular goal is a definite priority for near future.

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VITA

Sunayana Mitra was born on the 18th of November in the small town of Asansol, in West

Bengal state of India. As the nature of her father’s job in a nationalized bank required a lot of moving, she grew up in the Calcutta (now Kolkata), Asansol, Bombay (now Mumbai). She finished her High School studies from Kendriya Vidyalaya Ballyganj in Calcutta in 2002. After that she moved to Nagpur City situated in Maharashtra state for Undergraduate studies. She obtained her Bachelor’s degree in Science from Lady Amritbai Daga College for Women

(affiliated to Nagpur University) in 2005 and went on to pursue her a Master’s degree in Science from Hislop School of Biotechnology (HSB), Hislop College (also affiliated to Nagpur

University). She obtained her degree in 2007 majoring Biotechnology. After that she joined the staff of HSB as an instructor and taught Molecular Biology and Biochemistry to undergraduates for a semester, while preparing for obtaining admission to PhD. programs in the US.

She left her home country in fall of 2008 to join the Biochemistry PhD program at

Louisiana State University (LSU), offered by the Department of Biological Sciences. Due to circumstances out of her control, she had to leave her original laboratory and research project in

2011. She remained in the program and joined another laboratory and started her research afresh.

She successfully defended her thesis on the 25th of June 2015. She plans to pursue scientific research as a Postdoctoral Researcher in the near future.