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Graduate School
2015
Insights into 5-Lipoxygenase Active Site and
Catalysis
Sunayana Mitra
Louisiana State University and Agricultural and Mechanical College
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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
in
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)
ii
कर्मणयेवाधिकारस्ते र्ा फलेषु कदाचन।
र्ा कर्मफलहेतर्ु र्मभ ाम ते सङ्गोऽस््वकर्मधण।
(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.
vi
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 5LIPOXYGENASE................................................................................................................ 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-SHPETE, 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)
1
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.
2
Structurally, all lipoxygenases, irrespective of their origins, share a predominantly αhelical 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).
.
3
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
gene
Former
name
Mouse
gene
Former
name
Major
expression
References
ALOX5
5-LOX
alox5
alox5
Leukocytes,
macrophages,
dendritic cells
(Funk, Hoshiko et al.
1989, Ghosh 2003,
Faronato, Muzzonigro
et al. 2007, Wasilewicz,
Kolodziej et al. 2010)
ALOX12
pl12LOXb
alox12
pl12LOX
Thrombocytes,
skin
(Funk, Furci et al.
1990, Johnson, Brass et
al. 1998, Virmani,
Johnson et al. 2001)
ALOX12B
12RLOX
alox12b 12RLOX
Skin
(Boeglin, Kim et al.
1998, Meruvu, Walther
et al. 2005)
ALOX15
12/15LOX
alox15
Eosinophils,
bronchial
epithelium
(Sigal, Grunberger et
al. 1988, Sigal, Sloane
et al. 1993)
ALOX15B
15LOX2
alox15b 8-LOX
Hair roots,
skin, prostate
(Brash, Boeglin et al.
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)
alox12e elox12
Skin
(Funk, Chen et al.
2002)
Pseudogene
Lc12LOXa
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.
6
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 (5hydroperoxyeicosatetraenoic 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)-Sglutathyionyl-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 γ8
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.
9
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 Cterminal 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).
11
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.
15
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).
17
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.
18
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 15LOX-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 5LOX 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
22
(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 5lipoxygenase (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 5HETE 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
DGLA
X
X
X
X
AEA
X
X


NAGly
X



27
F177A
F177A
Y181A
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.
29
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.
30
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
Y181A
A603L
A603L
Y181A
W147L
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
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
Kinetic
parameters
Table 4b. Summary of product formation by ‘plug’ mutants.
Products
5-HETE
4.1±0.2
11.1±0.3
11-HETE
nd
nd
nd
nd
nd
0.57±0.02
nd
nd
nd
0.6±0.04
0.27±.04
nd
0.4±0.03
nd
nd
nd
nd
nd
nd
0.22±.04
nd
nd
1.9±.06
0.66±.05
4.1±0.5
0.6±.05
0.17±.01
0.25±0.1
8/12- HETE
15-HETE
“other”
5.8±0.3
nd- Not detected
31
3.1±0.4
0.13±0.1
7.7±0.03
1.7±0.6
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).
32
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.
33
Table 5. Summary of the different properties of His600 mutants
Mutant
Tm (°C)
St-5-LOX
H600V
H600A
F177A:Y181A:H
600V
53.1±0.1
56.4±
0.2
56.7±
0.2
54.8 (0.1)
Kinetic
parameters
Km (µM)
14±1.6
nd
27±0.7
5.4±0.5
Kcat (s-1)
Products
0.8±0.03
nd
<0.01
0.3±0.01
5-HETE
4.1±0.2
nd
8/12- HETE
15 HETE
“Other”
nd
nd
nd
nd
nd
3.4±.02
nd
0.6±0.04
nd
nd
nd
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.
34
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 5HETE 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 “headfirst” substrate entry.
37
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 Ushaped 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
38
(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 5LOX 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
39
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 Tyr181 are largely solvent accessible amino acids at the entrance to the site. However, the 8RLOX 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.
40
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.
41
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.
𝑉max is the maximal velocity, 𝑆 is the substrate concentration, 𝐾m is the Michaelis constant.
𝑣=
Equation 1
𝑉max∙[𝑆]
𝐾m+[𝑆]
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.
42
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.
43
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.
44
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.
46
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).
47
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.
48
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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.
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