structural studies of the klebsiella ......figure 23 michaelis-menten plot of reaction velocity vs....
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STRUCTURAL STUDIES OF
THE KLEBSIELLA PNEUMONIAE PANTOTHENATE KINASE
IN COMPLEX WITH
PANTOTHENAMIDE SUBSTRATE ANALOGUES
by
Buren Li
A thesis submitted in conformity with the requirements
for the degree of Master of Science.
Graduate Department of Pharmacology and Toxicology
University of Toronto.
© Copyright by Buren Li (2012)
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Structural studies of the Klebsiella pneumoniae pantothenate kinase in complex with
pantothenamide substrate analogues
Buren Li
Master of Science
2012
Department of Pharmacology and Toxicology
University of Toronto
ABSTRACT
N-substituted pantothenamides are analogues of pantothenate, the precursor of the
essential metabolic cofactor coenzyme A (CoA). These compounds are substrates of
pantothenate kinase (PanK) in the first step of CoA biosynthesis, possessing
antimicrobial activity against multiple pathogenic bacteria. This enzyme is an attractive
target for drug discovery due to low sequence homology between bacterial and human
PanKs. In this study, the crystal structure of the PanK from the multidrug-resistant
bacterium Klebsiella pneumoniae (KpPanK) was first solved in complex with N-
pentylpantothenamide (N5-Pan). The structure reveals that the N5-Pan pentyl tail is
located within a highly aromatic pocket, suggesting that an aromatic substituent may
enhance binding affinity to the enzyme. This finding led to the design of N-pyridin-3-
ylmethylpantothenamide (Np-Pan) and its co-crystal structure with KpPanK was solved.
The structure reveals that the pyridine ring adopts alternative conformations in the
aromatic pocket, providing the structural basis for further improvement of
pantothenamide-binding to KpPanK.
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ACKNOWLEDGEMENTS
First and foremost, I would like to extend my gratitude to my parents and sisters
for their unwavering love and support.
My time in the graduate program has been made easier and enjoyable because of
generous laboratory colleagues who are always willing to share their expertise and
knowledge. I would like to especially thank Dr. BumSoo Hong for all those hours we
spent troubleshooting my errors and of course, talking about life. I am also grateful to
Johnny Guan, who was my mentor when I first arrived at the Park lab and has
encyclopedic knowledge of all laboratory practices and techniques. It was also a pleasure
to have worked alongside fellow students Hanyoul Lee, Cathy Kim, Kathy Mottaghi,
Scott Hughes and Negar Nosrati. I would like to thank former members of the Park
group, Drs. Yufeng Tong, Nan Zhong, as well as Lucy Nedyalkova, Slav Dimov and
Limin Shen, who have never hesitated to lend a hand in my times of need.
All work presented in this thesis was performed at the Structural Genomics
Consortium (SGC), a truly ideal environment for structural biology research. I am
indebted to Dr. Wolfram Tempel for helping me with crystal screening and Synchrotron
data collection, and to Drs. Guillermo Senisterra and Abdellah Al-Hassani for valuable
technical assistance in running kinetic assays. I would also like to extend my thanks to
Drs. David Smil and Yuri Bolshan, the chemists at the SGC who generously provided the
compounds used in these studies.
I have also benefitted from the kindness and expertise of my co-supervisor Dr.
Peter McPherson and advisor Dr. David Riddick, both of whom agreed to serve in their
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respective capacities without hesitation. They have my thanks for going above and
beyond what I expected whenever I consult with them.
I would also like to thank my defense committee members: Dr. Martin Zack
(chair), Dr. Jeffrey Lee (external appraiser), Dr. Hong-Shuo Sun (internal appraiser) and
Dr. David Riddick (additional voting member). Their careful review of this thesis is
greatly appreciated.
Last but definitely not least, I would like to extend my sincerest thanks to my
supervisor Dr. Hee-Won Park. I feel extremely fortunate to have met such a bighearted,
inspiring and selfless mentor. The rewarding journey wasn’t always smooth, and results
didn’t always come readily. But I would always be reassured by Dr. Park that with hard
work and strong convictions, things will work out.
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TABLE OF CONTENTS
Abstract ii
Acknowledgements iii-iv
Table of Contents v-vi
Lists of Tables and Appendix vii
List of Figures viii-ix
Abbreviations x-xi
1. INTRODUCTION
1.1 Urgency for antimicrobial drug discovery 1-3
1.2 Pantothenate Essentiality and Uptake Mechanisms 3-5
1.3 Overview of Coenzyme A 5-11
1.4 Synthesis of Coenzyme A 11
1.4.1 De novo Pantothenate Synthesis 12
1.4.2 Coenzyme A Synthesis from Pantothenate 14
1.4.2.1 Conversion of Pantothenate to 4’-phosphopantothenate 16
1.4.2.2 Conversion of 4’-phosphopantothenate to 4’-
phosphopantetheine
16-17
1.4.2.3 Conversion of 4’-phosphopantetheine to coenzyme A 17
1.5 Pantothenate Kinase as point of drug discovery 17-18
1.5.1 Pantoyltaurine 20
1.5.2 N’-pantoyl-substituted amide 20-21
1.5.3 N-substituted pantothenamide 21-22
1.6 Overview of Pantothenate Kinases 24
1.6.1 Type I Pantothenate Kinases 24-27
1.6.2 Type II Pantothenate Kinases 27-31
1.6.3 Type III Pantothenate Kinases 31-32
1.7 Hypothesis and Rationale for Study 38
1.7.1 Aims and Approaches 38-39
1.7.2 Rationale for Experimental Approach
1.7.2.1 Structure Determination of Macromolecules 39
1.7.2.2 X-ray Crystallography 39-40
1.7.2.3 Protein Crystallization 40
1.7.2.4 Data Collection 42
1.7.2.5 Structure Determination 42-43
2. MATERIALS AND METHODS
2.1 Materials 43-44
2.2 Methods
2.2.1 Preparation of Expression Plasmid 46
2.2.2 Protein Expression and Purification 49-50
2.2.3 Protein Crystallization and Data Collection 52-53
2.2.4 Structure Determination, Refinement and Validation 57-59
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2.2.5 Spectrophotometric Assessment of Substrate Kinetics 63
3. RESULTS
3.1 Structural Overview of KpPanK 65
3.1.1 Nucleotide-binding site 65-66
3.1.2 N5-Pan binding site of KpPanK 69
3.1.3 Np-Pan binding site of KpPanK 71-72
3.2 KpPanK substrate kinetics 74
4. DISCUSSION
4.1 Comparison with EcPanK 77-78
4.2 Comparison with MtPanK 80-81
4.3 Modeling of a Branched Compound 84-85
4.4 KpPanK Substrate Kinetics 88
4.5 Summary of Findings 88-89
4.6 Recommendations for Future Studies 89-92
References 93-100
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LIST OF TABLES
Table I Sequences of primers used to generate each KpPanK construct. 48
Table II Summary of substrates used for KpPanK co-crystallization and the
best resolution achieved.
56
Table III Data collection and refinement statistics for KpPanK crystals. 61
Table IV Characterization of KpPanK substrate kinetics. 76
Table V Summary of polar interactions involving the pantothenate moiety of
substrates in KpPanK, EcPanK and MtPanK structures.
82
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LIST OF FIGURES
Figure 1 Chemical structure of coenzyme A. 7
Figure 2 Overview of fatty acid synthesis. 9
Figure 3 De novo pantothenate biosynthesis pathway in bacteria. 13
Figure 4 CoA biosynthesis from pantothenate in bacteria. 15
Figure 5 Chemical structures of pantothenate and related derivatives. 19
Figure 6 Proposed mechanisms of pantothenamide toxicity. 23
Figure 7 Phylogenetic distributions of prokaryotic and eukaryotic
pantothenate kinases from notable organisms.
33
Figure 8 Sequence-based alignments of prokaryotic and eukaryotic PanKs
from types I (A), II (B), and III (C).
34-36
Figure 9 Comparison of the structures and dimer folds of types I, II and III
bacterial PanKs.
37
Figure 10 Phase diagram of crystallization. 41
Figure 11 Overview of the pET28-MHL expression vector. 45
Figure 12 Small scale test of expression of KpPanK constructs. 47
Figure 13 Purification of KpPanK. 51
Figure 14 Crystals of KpPanK co-crystallized with N5-Pan. 54
Figure 15 Crystals of KpPanK co-crystallized with Np-Pan. 55
Figure 16 Diffraction patterns of KpPanK crystals. 60
Figure 17 Matthews Probability calculation of the oligomeric state of the
KpPanK asymmetric unit.
62
Figure 18 Pyruvate kinase (PK)/lactate dehydrogenase (LDH) coupled assay
for characterization of kinase activity.
64
Figure 19 Structure of a KpPanK subunit. 67
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Figure 20 Interaction of KpPanK nucleotide-binding residues with ADP. 68
Figure 21 Residues of the KpPanK substrate-binding site. 70
Figure 22 Interactions of the pyridine of Np-Pan with substrate pocket
residues.
73
Figure 23 Michaelis-Menten plot of reaction velocity vs. substrate
concentration.
75
Figure 24 Structural differences between KpPanK and EcPanK substrate
binding sites.
79
Figure 25 Comparison of the substrate-binding sites of KpPanK and MtPanK. 83
Figure 26 Modeling of a branched version of Np-Pan in the KpPanK substrate-
binding site.
86
Figure 27 Modeling of a branched derivative of Np-Pan in human PanK3. 87
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ABBREVIATIONS
ACP = acyl carrier protein
ACS = acetyl-CoA synthetase
AnPanK = Aspergillus nidulans pantothenate kinase
ASKHA = acetate and sugar kinase/heat shock protein 70/actin
Baf = Bvg accessory factor
DPC = dephospho-coenzyme A
DPCK = dephospho-coenzyme A kinase
EcPanK = Escherichia coli pantothenate kinase
Ed-CoA = ethyldethia-CoA
ESBL = extended spectrum β-lactamase
FAS = fatty acid synthase
hPanK3 = human pantothenate kinase isoform 3
IPTG = isopropyl β-D-1-thiogalactopyranoside
MIC = minimum inhibitory concentration
mPanK = Mus musculus pantothenate kinase
MR = molecular replacement
MtPanK = Mycobacterium tuberculosis pantothenate kinase
N5-Pan = N-pentylpantothenamide
N7-Pan = N-heptylpantothenamide
N9-Pan = N-nonylpantothenamide
Np-Pan = N-pyridin-3’-ylmethylpantothenamide
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PanF = pantothenate permease
PanK = pantothenate kinase (coaA)
P-Pan = 4’-phosphopantothenate
PP = 4’-phosphopantetheine
PPAT = phosphopantetheine adenyltransferase (coaD)
PPC = phosphopantothenoylcysteine
PPCDC = phosphopantothenoylcysteine decarboxylase (coaC)
PPCS = phosphopantothenoylcysteine synthetase (coaB)
RMSD = root mean square deviation
SVMT = sodium-dependent multi-vitamin transporter
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1. INTRODUCTION
1.1 Urgency for antimicrobial drug discovery
Drug-resistant pathogens represent a major challenge to healthcare and drug
development. Conventional classes of antibiotics that were once capable of controlling
infections are becoming more and more ineffective (Rice 2012). The rate of drug
development has not been able to keep up with the increasing number of therapeutic
options lost because of drug resistance (Bassetti, Ginocchio et al. 2011). Most
antimicrobial agents share conventional cellular targets that include interfering with cell
wall formation, membrane function, and DNA and protein synthesis (Neu 1989; Rice
2012). Under selective pressures introduced through excessive use of antibiotics,
microorganisms have developed resistance to drugs by: increased efflux, alteration of the
drug targets, and enzymatic inactivation (Neu 1989).
Resistance in gram-negative bacterial pathogens is particularly troubling; their
lipopolysaccharide outer membrane provide intrinsic resistance against several classes of
antibiotics, such as macrolides and cationic peptides (Delcour 2009). As such, there are
limited treatment regimens for infection caused by gram-negative bacteria, which in some
cases are prompt recipients of resistance genes. A prominent example is the acquisition
of extended spectrum β-lactamases (ESBL) in Enterobacteriaceae that hydrolyze a broad
range of penicillins, and render numerous members of the drug class ineffective (Paterson
and Bonomo 2005). The first ESBL discovered was TEM-1 (named after Temoniera, the
source patient) (Bradford 2001). A second related enzyme was discovered and named
TEM-2. A third, unrelated and much less common ESBL is SHV, named so due to the
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variable effects sulfhydryl compounds had on substrate specificity. The advent of
cephalosporins was considered a major breakthrough in countering β-lactamase-mediated
drug resistance (Paterson and Bonomo 2005). Soon after, overuse of these drugs led to
the emergence of ESBLs capable of hydrolyzing cephalosporins; mutations that promote
substrate promiscuity are found in the genes that encode the three ESBLs (Philippon,
Labia et al. 1989). Recently, there has been a rise in bacteria that produce
carbapenemases, a β-lactamase-like enzyme that provides resistance to carbapenem drugs
(often considered drugs of last resort) (Daikos and Markogiannakis 2011). Widespread
drug resistance can result in treatment failure and increased mortality (Tumbarello, Spanu
et al. 2006). Therefore, the development of new drugs with novel mechanisms of action
and/or cellular targets is crucial to treat increasingly drug-resistant infections and
alleviate a depleted drug pipeline.
Klebsiella pneumoniae is a prominent gram-negative and drug-resistant
bacterium. Pathogenic strains are typically expressors of ESBLs (belonging to the TEM
and SHV classes) and display resistance to a wide spectrum of beta-lactams including
many penicillins and cephalosporins (Paterson, Hujer et al. 2003). Infections caused by
multi-drug resistant strains of K. pneumoniae are mainly treated with carbapenems (e.g.
imipenem and meropenem) (Yigit, Queenan et al. 2001). This therapeutic option is
becoming less viable, with the increasing findings of carbapenem-resistant K.
pneumoniae isolates; these strains display lowered drug permeability due to altered porin
protein (Ardanuy, Linares et al. 1998) and/or expression of the AmpC carbapenemase
(Bradford, Urban et al. 1997). Carbapenem resistance in gram-negative bacteria
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underscores the urgent need for novel drug discovery, considering the drugs’ status as
“agents of last resort” (Hirsch and Tam 2010).
1.2 Pantothenate Essentiality and Uptake Mechanisms
Williams et al. first discovered pantothenic acid (vitamin B5, its conjugate base is
called pantothenate) as a growth stimulant of Saccharomyces cerevisiae (Williams,
Lyman et al. 1933). Because of the ubiquitous nature of the acidic substance, it was
named after the Greek word pantothen, which means “from everywhere” (Williams,
Lyman et al. 1933). Insights into the chemical structure of pantothenic acid followed the
discovery of β-alanine as another yeast growth factor (Williams and Rohrman 1936). β-
alanine is a cleavage product of pantothenic acid, and yeast excretes excess pantothenic
acid only when β-alanine is supplemented in the growth medium (Weinstock, Mitchell et
al. 1939).
Snell et al. found that extracts from pig liver and yeast share a growth factor
essential for the survival of lactic acid bacteria such as Lactobacillus delbruckii (Snell,
Strong et al. 1937). Purification and chemical characterization of this unknown substance
led to its identification as pantothenic acid (Snell, Strong et al. 1938; Snell, Strong et al.
1939). Pantothenic acid can also stimulate the growth of bacterial pathogens such as
Corynebacterium diphtheriae (Evans, Handley et al. 1939); β-alanine is also a growth
factor at higher concentrations (Mueller and Cohen 1937). In other bacteria such as
Scenedesmus obliquus, the amino acid precursor cannot be substituted for the essential
vitamin (Algeus 1951). The synthetic pathway of pantothenate was first discovered and
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characterized in Escherichia coli (Merkel and Nichols 1996). Despite disruption of any
one of the enzymes in the pantothenate synthesis pathway, E. coli is viable as long as
pantothenate is present in the medium (Gerdes, Scholle et al. 2002). A racemic mixture
of pantothenic acid possesses 50% activity of the dextrorotatory (D) isomer, while the
levorotatory shows none (Stiller, Harris et al. 1940). Pantothenate was later discovered to
be the precursor of the essential coenzyme A (CoA) metabolic cofactor (described below)
(Hoagland and Novelli 1954).
The uptake of pantothenate occurs by means of a transporter present in virtually
all bacteria (Gerdes, Scholle et al. 2002; Genschel 2004). In E. coli, exogenous
pantothenate is readily taken up by a 12-transmembrane transporter called pantothenate
permease (PanF), encoded by the PanF gene (Jackowski and Alix 1990). The activity of
PanF relies on a sodium ion gradient and has a Kt (transporter constant, analogous to
Michaelis-Menten constant) of 0.4μM for pantothenate. Over 90% of pantothenate is
trapped by phosphorylation within 5 minutes of entry (Jackowski and Alix 1990). While
an increase in PanF expression results in increased intracellular pantothenate, there is no
corresponding relationship in levels of the final product CoA (Vallari and Rock 1985). In
addition, the permease also possesses pantothenate efflux activity (Vallari and Rock
1985). The E. coli PanF shares some similarity in sequence to the E. coli proline
symporter as well as mammalian glucose transporters (Reizer, Reizer et al. 1990). These
transporters share two conserved tyrosine residues that are proposed to be essential for
binding Na+ ions.
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In chicks and rats, the discovery that a sodium-dependent, secondary active
process was responsible for pantothenate uptake was the first evidence of the vitamin’s
uptake in mammals (Fenstermacher and Rose 1986). In humans, pantothenate is
transported into the cytosol by the sodium-dependent multi-vitamin transporter (SVMT)
(Prasad, Wang et al. 1999). The water-soluble vitamins biotin and lipoate are also
substrates for the SVMT transporter (Prasad, Wang et al. 1998). The transport of the
vitamins is dependent on both a sodium gradient and a specific membrane potential
(Prasad and Ganapathy 2000). The vitamin lipoate is capable of inhibiting the uptake of
the other two vitamins (Prasad, Wang et al. 1998). The Kt values of this transporter for
pantothenate and biotin are 1-3μM, and slightly higher for lipoate at 8-20μM (Prasad,
Wang et al. 1999).
1.3 Overview of Coenzyme A
During the investigation of a detoxification reaction in liver extract, Lipmann
discovered a cofactor that is necessary for the acetylation of aromatic amines; the
substance was thus termed coenzyme A (CoA, A for acetylation) (Lipmann, Kaplan et al.
1947). CoA is also required for acetylating other substances such as choline, histamine,
amino acids and glucosamine (Lipmann 1953). The chemical structure of CoA
comprises 3’-adenosine diphosphate, pantothenate and β-mercaptoethylamine moieties
(Fig. 1); the latter two constitute a pantetheine group (Baddiley, Thain et al. 1953). The
knowledge that CoA is synthesized from pantothenate and is required for acetylation led
to investigations into the effects of pantothenate-deficient conditions in rats; not
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unexpected, the ability of rats to carry out acetylation was greatly diminished, but rapidly
restored when pantothenate is readministered (Snell and Wright 1950). Lipmann also
found a correlation between CoA and lipid levels, as lipid contents in rat liver and yeast
are lower in CoA-poor conditions (Lipmann 1953).
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Figure 1. Chemical structure of coenzyme A. CoA is made up of 3’-adenosine
diphosphate, pantothenate and β-mercaptoethylamine moieties, the last two of which
constitute a pantetheine group.
3’-adenosine
diphosphate pantothenate β-mercapto-
ethylamine
pantetheine
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CoA is a universally conserved acyl group carrier essential in multiple
physiological processes that include Claisen condensation reactions and the citric acid
cycle (also known as Kreb cycle, and tricarboxylic acid cycle). Claisen condensation is
the formation of carbon-carbon bonds between two esters, or one ester and a carbonyl
compound, of which fatty acid synthesis is a notable example (Heath and Rock 2002).
The essentiality of CoA to fatty acid synthesis is two-fold. Firstly, CoA is a
precursor for acyl carrier protein (ACP), an essential component of the fatty acid synthase
(FAS) complex. Holo-ACP synthase converts apo-ACP to holo-ACP (the active form)
by transferring the phosphopantetheine moiety from CoA onto the serine 36 side chain
hydroxyl of apo-ACP (Flugel, Hwangbo et al. 2000). Besides synthesizing ACP, acyl
groups derived from acyl-CoA are required to activate/prime components of the FAS
complex. First, the acetyl group from acetyl-CoA is transferred onto a cysteine residue of
the FAS complex. Similarly, the phosphopantetheine of holo-ACP is charged with
malonyl from malonyl-CoA. Fatty acid synthesis then proceeds through repeated cycles
of condensation, reduction, dehydration and isomerization steps whereby the fatty acid
chain is extended two carbon units at a time by malonyl groups delivered by CoA (Fig. 2)
(Lehninger 2004). In conditions of CoA deficiency, decreased levels of saturated and
unsaturated fatty acids are observed in E. coli (Jackowski and Rock 1986).
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Figure 2. Overview of fatty acid synthesis. The FAS complex first
receives acetyl (cysteine) and malonyl
(ACP pantetheine) groups that are
delivered by CoA.
1. The condensation step involves
transfer of the acetyl group to the
ACP malonyl group; the CH2 of
malonyl nucleophilically attacks the
carbonyl carbon of the acetyl group.
The reaction is driven by the highly
exergonic acyl bond cleavage of
decarboxylation.
2. In the reduction step, the β carbonyl
is reduced using the electron-
donating cofactor NADPH.
3. Water is removed in an elimination
reaction between the second and
third carbon units.
4. In a second reduction step, the
double bond is reduced to yield a
saturated bond.
5. To prepare for a new cycle, the
newly formed acyl group is
transferred to the FAS cysteine, and
the ACP pantetheine receives a new
malonyl group from malonyl-CoA.
(from Principles of Biochemistry 4e.
Lehninger 2004. Figure 21-2)
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Acetyl-CoA (synthesized from acetate and CoA), the most common esterified
CoA derivative, is central to cellular metabolism (Lehninger 2004). In the citric acid
cycle oxaloacetate is acetylated using acetyl-CoA to generate citrate. Each cycle
generates the reduced coenzymes NADH and FADH2 that contribute to oxidative
phosphorylation in ATP synthesis, accounting for over 90% of cellular energy
requirements. In addition, the citric acid cycle generates precursors of amino acids and
nucleotides, such as oxaloacetate and α-ketoglutarate (Lehninger 2004). In E. coli, a
consequence of CoA depletion is overall deficiency in protein synthesis; this is likely due
in part to a lack of succinyl-CoA suggesting that amino acid precursors generated by the
citric acid cycle are insufficient to support amino acid synthesis (Jackowski and Rock
1986).
Acetylation plays diverse regulatory roles in prokaryotes. In E. coli, the RimL
acetyltransferase uses acetyl-CoA to acetylate L12 ribosomal stock proteins, which
increases the level of interaction within the stock complex to enhance stability in
conditions of stress (Tanaka, Matsushita et al. 1989; Gordiyenko, Deroo et al. 2008). It is
possible that protein acetylation serves as a signal for degradation, similar to eukaryotic
proteolysis (Hwang, Shemorry et al. 2010). Protein lysine acetylation in bacteria is
essential for multiple biochemical pathways that include transcription, translation, protein
folding, and amino acid and nucleotide biosynthesis (Jones and O'Connor 2011). In E.
coli, proteins that are lysine-acetylated catalyze reactions in glycolysis, the citric acid
cycle as well as carbohydrate metabolism (Yu, Kim et al. 2008). In Salmonella enterica,
acetyl-CoA is a negative feedback regulator of its own synthesis by contributing to the
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acetylation of an acetyl-CoA synthetase (ACS) lysine residue to block ATP-dependent
adenylation of acetate; the sirtuin CobB activates ACS via deacetylation (Starai, Celic et
al. 2002). Also in S. enterica, reversible acetylation helps to regulate metabolism by
modifying enzymes involved in gluconeogenesis and glycolysis in response to specific
carbon sources (Wang, Zhang et al. 2010). As it turns out, approximately 90% of
enzymes involved in metabolism are acetylated, and the overall level of acetylation in
carbon source-reponsive proteins is significantly higher when cells are grown in glucose
versus citrate (Wang, Zhang et al. 2010). These data suggest that acetyl-CoA, a
metabolic molecule itself, is used to regulate metabolic homeostasis (Wang, Zhang et al.
2010). Bacteria also possess acetyltransferases that catalyze acetyl-CoA-dependent
acetylation and inactivation of aminoglycoside antibiotics, contributing to a significant
global rise in aminoglycoside resistance (Vetting, Magnet et al. 2004).
1.4 Synthesis of Coenzyme A
The synthesis of CoA can be separated into two parts: de novo synthesis of
pantothenate, and the synthesis of CoA from pantothenate (Begley, Kinsland et al. 2001).
The first pathway is limited to fungi, plants and certain bacteria; mammals, including
humans, must depend on diet in obtaining pantothenate (Raman and Rathinasabapathi
2004). The latter pathway is essential and conserved in all living systems (Genschel
2004).
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1.4.1 De novo Pantothenate Synthesis
Pantothenate synthesis was first characterized in E. coli, and occurs in 4
enzymatic steps (Fig. 3) (Merkel and Nichols 1996). First, α-ketoisovalerate is
hydroxymethylated at the α-carbon position by ketopantoate hydroxymethyltransferase
(KPHMT, encoded by the panB gene) to yield ketopantoate (Merkel and Nichols 1996).
Ketopantoate is then reduced at its carbonyl oxygen to hydroxyl by NADPH-dependent
ketopantoate reductase (KPR, encoded by panE gene) to produce pantoate (Frodyma and
Downs 1998). β-alanine, derived from the decarboxylation of L-aspartate by aspartate
decarboxylase (ADC, encoded by panD gene), is then combined with pantoate in an
ATP-dependent condensation reaction catalyzed by pantothenate synthetase (PS, encoded
by the panC gene) to produce pantothenate (Merkel and Nichols 1996).
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Figure 3. De novo pantothenate biosynthesis pathway in bacteria. Enzymes are
abbreviated as follows: KPHMT (ketopantoatehydroxymethyl transferase), KPR
(ketopantoate reductase), PS (pantothenate synthetase), ADC (aspirate decarboxylase)
(from Genschel et al., 2004).
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1.4.2 Coenzyme A Synthesis from Pantothenate
CoA synthesis from the pantothenate occurs in 5 enzymatic steps (Fig. 4). In the
first step, pantothenate kinase (PanK) catalyzes the phosphorylation of pantothenate.
Next, 4’-phosphopantothenate is conjugated with a cysteine residue by 4’-
phosphopantothenoyl cysteine synthetase, followed by decarboxylation by 4’-
phosphopantothenoyl cysteine decarboxylase to produce 4’-phosphopantetheine. The
adenylation of 4’-phosphopantetheine is catalyzed by 4’-phosphopantetheine
adenyltransferase to produce dephospho-CoA, which is phosphorylated by dephospho-
CoA kinase to produce CoA.
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Figure 4. CoA biosynthesis from pantothenate in bacteria. (from Genschel et al.,
2004).
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1.4.2.1 Conversion of Pantothenate to 4’-phosphopantothenate
The first step in CoA synthesis is the ATP-dependent phosphorylation of
pantothenate by pantothenate kinase (PanK, also known as coaA, encoded by the coaA
gene) to yield 4’-phosphopantothenate (P-Pan) (Jackowski and Rock 1981). This thesis
will focus on the structure and kinetic properties of the pantothenate kinase from the
bacterium Klebsiella pneumoniae. The kinetic, regulatory and structural properties of the
various classes of PanKs will be discussed in detail in a later section.
1.4.2.2 Conversion of 4’-phosphopantothenate to 4’-phosphopantetheine
In bacteria, such as E. coli, the synthesis of 4’-phosphopantetheine (PP) is
synthesized in two steps from P-Pan. The two enzymatic reactions are catalyzed by the
bifunctional 4’-phosphopantothenoylcysteine (PPC) synthetase/decarboxylase (PPC-
S/DC), encoded by the coaBC gene (Strauss, Kinsland et al. 2001). First, cysteine is
combined with P-Pan in a condensation reaction that uses CTP and releases CMP and
diphosphate as products. In the second step, the same enzyme then catalyzes the
decarboxylation of PPC into 4’-phosphopantetheine (PP) (Strauss and Begley 2001).
PPCDC was first discovered as a product of the dfp gene, whose N-terminal
domain shares sequence similarities with EpiD peptidylcysteine decarboxylase proteins
(Kupke, Uebele et al. 2000). It was renamed to coaBC when the C-terminal domain of
dfp was found to also possess PPCS activity (Strauss, Kinsland et al. 2001; Kupke 2002).
In humans however, the same reactions are catalyzed by two separate enzymes, PPC-S
and PPC-DC (Manoj, Strauss et al. 2003). Another distinction from the bacterial
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pathway is that the human PPC-S enzyme uses ATP instead of CTP to catalyze cysteine
conjugation (Manoj, Strauss et al. 2003).
1.4.2.3 Conversion of 4’-phosphopantetheine to Coenzyme A
The penultimate step in CoA synthesis is the conversion of PP to 3’-dephospho-
CoA (DPC) by phosphopantetheine adenyltransferase (PPAT, encoded by coaD gene)
(Geerlof, Lewendon et al. 1999). This reversible reaction is ATP-dependent, and releases
pyrophosphate as a product (Geerlof, Lewendon et al. 1999). Dephospho-coenzyme A
kinase (DPCK, also known as CoA synthase, and encoded by coaE gene) catalyzes the
ATP-dependent, final reaction in CoA synthesis by phosphorylating the 3’-hydroxyl
group of the ribose moiety to yield CoA (Mishra, Park et al. 2001). In eukaryotes
however, these two reactions are catalyzed by a two-domain protein that possesses both
catalytic activities (Zhyvoloup, Nemazanyy et al. 2002).
1.5 Pantothenate Kinase as a point of drug discovery
CoA biosynthesis is known to be universally essential, even in pathogenic
bacteria and fungi. Within this pathway, PanK is a practical target for drug discovery,
given that it catalyzes the rate-determining step in CoA biosynthesis (Vallari, Jackowski
et al. 1987). Inhibitor design based on CoA, a negative feedback regulator of its own
synthesis, is impractical, since CoA and its analogues cannot freely cross the bacterial
cell membrane (Mishra and Drueckhammer 2000). There is low sequence and structural
homology between prokaryotic and eukaryotic PanKs (Genschel 2004; Ivey, Zhang et al.
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18
2004; Hong, Yun et al. 2006; Hong, Senisterra et al. 2007). Most currently available
antibiotics have an intracellular target and must overcome the obstacles to entry presented
by the bacterial cell wall (Delcour 2009). Pantothenate and its derivatives (Fig. 5), from
a structural perspective, are virtually indistinguishable and are readily taken up by
bacteria via PanF transporters (Vallari and Rock 1985; Strauss and Begley 2002; Zhang,
Frank et al. 2004). The sections below outline pantothenate analogues that possess
antimicrobial activity.
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19
Figure 5. Chemical structures of pantothenate and related derivatives. A.
Pantothenate (The atom positions are labeled in the chemical structure). B.
Pantoyltaurine. C. N-pantoyl-substituted amine. D. N-substituted Pantothenamide. The
compounds differ in the carboxylic acid terminal (right side).
A. B.
C. D.
C1 C2
C3 C4
C6
N5 C7
C8
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20
1.5.1 Pantoyltaurine
The first pantothenate analogue discovered to show antibacterial activity is
pantoyltaurine, in which the carboxyl group of pantothenate in the C8 position is replaced
with a sulphonate group (Fig. 5B). Snell demonstrated the growth inhibitory effects of
pantoyltaurine on the lactic acid bacterium Lactobacillus arabinosus, and the addition of
pantothenic acid antagonizes the effect observed (Snell 1941). Pantoyltaurine is also
capable of inhibiting the growth of other pathogenic bacteria such as the streptococci S.
hemolyticus and S. pneumoniae (McIlwain 1942). Similarly the addition of pantothenate
to the growth medium provides some resistance to pantoyltaurine. In Corynebacterium
diphtheriae, pantoyltaurine shows differential growth inhibition depending on the
specific strain tested (McIlwain and Hawking 1943). In bacteria capable of de novo
pantothenate synthesis such as Escherichia coli, Proteus morgani and Staphylococcus
aureus, pantoyltaurine has no growth inhibitory effects (McIlwain and Hawking 1943).
Pantoyltaurine can be used to treat mice and rats infected with sulfonamide-resistant
strains of S. hemolyticus (McIlwain and Hawking 1943). A proposed mechanism of
action of pantoyltaurine is that its structural similarity to pantothenate leads to inhibition
of pantothenate-dependent cellular pathways (McIlwain 1942).
1.5.2 N-pantoyl-substituted amides
Pantothenol (Fig. 5C) (the terminal carboxyl of pantothenate is replaced by a
hydroxyl), is capable of inhibiting the growth of lactic acid bacteria that are incapable of
synthesizing pantothenate (Shive and Snell 1945). Like pantoyltaurine, pantothenol does
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21
not have any effect on the growth of either E. coli or S. aureus, and its antimicrobial
activity is likely due to its competitive inhibition of pantothenate. Similar to
pantothenate, only the dextrorotatory isomer possesses activity. Pantothenol also has
comparable potency to pantoyltaurine (Shive and Snell 1945). In contrast to bacteria,
pantothenol can promote growth in chicks with nearly equivalent efficacy as pantothenate
(Hegsted 1948).
1.5.3 N’-substituted pantothenamides
Previous pantothenate analogues have been synthesized via substitution of the
terminal carboxylic acid group. The addition of chemical moieties beyond the C8
position of pantothenate was explored in the form of pantothenamides. While showing
cytotoxicity in various bacteria, these compounds are especially effective against E. coli
(Clifton, Bryant et al. 1970) and S. aureus (Virga, Zhang et al. 2006).
In E. coli, N5-Pan is a substrate of PanK, and its phosphorylated product is
processed by most downstream enzymes to produce the CoA analogue ethyldethia-CoA
(Ed-CoA) (Strauss and Begley 2002). Part of the potency of N5-Pan against E. coli is
attributed to its rapid conversion to Ed-CoA, approximately 10.5 times faster than the
conversion of pantothenate to CoA (Strauss and Begley 2002). As efficient alternate
substrates of PanK, pantothenamides are effective competitive inhibitors of pantothenate
showing IC50 values below 60μM (Ivey, Zhang et al. 2004). CoA analogues derived
from pantothenamides lack the crucial terminal sulfhydryl group required for formation
of acyl-CoA thioesters and likely interfere with CoA-utilizing enzymes (Strauss and
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22
Begley 2002). Furthermore, in E. coli analogues of holo-ACP can be made from Ed-
CoA, and the accumulation of the inactive modified ACP molecules can lead to the
inhibition of fatty acid synthesis (Zhang, Frank et al. 2004). However, the notion that the
accumulation of inactive ACP underlies pantothenamide antimicrobial activity has been
challenged since ACP phosphodiesterase can readily hydrolyze the inactive ACP back to
apo-ACP (Thomas and Cronan 2010). In addition, exogenously supplementing fatty
acids to S. pneumoniae cannot provide full resistance to N5-Pan (Zhang, Frank et al.
2004). Compared with untreated E. coli cells, the pool of intracellular acetyl-CoA is
significantly reduced upon treatment with N5-Pan, which points to the inhibition of CoA
synthesis as an underlying mechanism of pantothenamide toxicity (Thomas and Cronan
2010). The proposed mechanisms of action of pantothenamides in E. coli are illustrated
in Figure 6.
In S. aureus, the mechanism of action of pantothenamides is unclear.
Pantothenamides were reported to inhibit PanK activity in S. aureus in contrast to being
pseudosubstrates in E. coli (Choudhry, Mandichak et al. 2003). However, exposing
strains of S. aureus to pantothenamides can lead to accumulation of inactivated ACP and
deficient fatty acid levels, suggesting that S. aureus and E. coli are probably inhibited by
the same mode of action (Virga, Zhang et al. 2006).
First-generation pantothenamides are capable of interfering with the growth of
human cells. When tested in human HepG2 cells these compounds showed a significant
level of growth inhibition with IC50 (concentration required to inhibit growth by 50%)
values as low as 64 and 128μg/mL (Choudhry, Mandichak et al. 2003).
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23
Figure 6. Proposed mechanisms of pantothenamide toxicity in E. coli.
The chemical structures of pantothenate, CoA and ACP are shown on the left. The
corresponding structures of N5-Pan and its downstream products are shown on the right.
Green and red arrows represent inductive/stimulatory and inhibitory effects, respectively.
The orange arrows indicate the absence of the essential sulfhydryl group essential for the
biological function of carrying acyl groups. (adapted from Thomas and Cronan, 2010).
.
Pan
CoA
ACP
N5-Pan
N5-CoA
N5-ACP Fatty Acid Synthesis
CoA-utilizing pathways
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24
1.6 Overview of Pantothenate Kinases
PanK catalyzes the first step of CoA biosynthesis by catalyzing the ATP-
dependent phosphorylation of the precursor pantothenate. PanKs are divided into three
classes based on amino acid sequence, structure, regulatory properties and substrate
kinetics (Fig 7). Type I PanKs are the first PanKs discovered and characterized,
predominantly found in prokaryotes. Type II PanKs are mainly found in eukaryotic
species, but interestingly also in select bacteria. Type III PanKs have an even wider
distribution within the bacterial kingdom compared with type I enzymes. The sections
below outline the properties distinct to each class.
1.6.1 Type I Pantothenate Kinases
The Escherichia coli PanK (EcPanK) is the best characterized type I enzyme.
The enzyme is encoded by the coaA gene, which when translated gives two products of
molecular weight 36.4kDa and 35.4 kDa (a difference of 8 N-terminal residues) (Song
and Jackowski 1992). In E. coli, a concentration 8μM β-alanine in the extracellular
medium results in maximal CoA intracellular concentrations (Jackowski and Rock 1981).
Higher β-alanine concentrations produce an amount of non-phosphorylated pantothenate
more than that required to maintain an optimal CoA level, leading to pantothenate
excretion (Jackowski and Rock 1981). Furthermore, strains harboring multiple copies of
the coaA gene express 76-fold higher levels of EcPanK, but only produce 2.7-fold higher
levels of CoA (Song and Jackowski 1992). These findings suggest that EcPanK plays a
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25
key regulatory role in CoA biosynthesis (Jackowski and Rock 1981; Song and Jackowski
1992).
EcPanK exists as a homodimer in solution (Song and Jackowski 1994). The
enzyme contains the Walker A phosphate-binding motif (GXXXXGKS) and belongs to
the P-loop kinase superfamily (Walker, Saraste et al. 1982; Yun, Park et al. 2000).
Kinetic studies have revealed sequential substrate binding in EcPanK; the binding of ATP
is required for binding of pantothenate (Song and Jackowski 1994). The binding of ATP
to one subunit of an EcPanK dimer promotes positive cooperative ATP-binding to the
second subunit (Song and Jackowski 1994). Kinetic characterization reveals that the
Michaelis-Menten constants (Km) for pantothenate and ATP are 36 and 136μM,
respectively.
In line with its presumed regulatory role in CoA biosynthesis, EcPanK is
negatively regulated by feedback inhibition with CoA (Vallari, Jackowski et al. 1987).
Non-acylated CoA inhibits EcPanK activity approximately five times more potently than
esterified derivatives like acetyl-CoA. CoA can also competitively inhibit the binding of
ATP (Vallari, Jackowski et al. 1987). A lysine residue of the P loop is essential for both
CoA and ATP binding; the lysine(101)-methionine mutant cannot not bind either
compound (Song and Jackowski 1994).
The structures of EcPanK in complex with non-hydrolyzable ATP analogue
AMPPNP and CoA are available (PDB: 1ESM and PDB: 1ESN) (Yun, Park et al. 2000).
EcPanK is a dimer in the asymmetric unit. Comparison of the two co-crystal structures
reveal that the α, β phosphates of CoA and the β, γ phosphates of AMPPNP occupy the
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26
same space in the active site, and provides structural basis for CoA inhibition of EcPanK.
Specifically, the biphosphates compete for binding to lysine 101 (Yun, Park et al. 2000).
Interestingly the adenine moiety of CoA does not occupy the same space as that of
AMPPNP, but instead flips to occupy another protein cleft (Yun, Park et al. 2000). The
CoA-bound structure also reveals the basis for more potent inhibition by CoA compared
with its thioesters; the terminal thiol group of CoA is located within a confined pocket in
which acyl groups of the CoA thioesters cannot optimally fit (Yun, Park et al. 2000).
Comparison of the two co-crystal structures also reveals three key residues involved in
CoA binding, but not ATP binding. This finding is confirmed by mutations of Arg106,
His177 and Phe247 to alanine, which reveal decreased potency of CoA inhibition while
retaining catalytic activity (Rock, Park et al. 2003). E. coli strains expressing these
mutants show significantly higher intracellular levels of phosphorylated pantothenate
derivatives and CoA, providing further evidence of EcPanK’s key regulatory role in CoA
synthesis (Rock, Park et al. 2003).
The ternary complex structure of EcPanK bound with ADP and pantothenate is
also available (Fig. 9A) (PDB: 1SQ5) (Ivey, Zhang et al. 2004). When superimposed
onto the EcPanK-AMPPNP complex, the overall protein fold of the ternary complex is
conserved with the exception of significant movement of a loop region containing
residues 243-263; this stretch of residues is thought to act as a lid that closes over the
active site upon substrate-binding. Superimposition with the EcPanK-CoA complex
reveals that pantothenate of the ternary complex and the pantetheine moiety of CoA have
the same mode of binding. The ternary complex was also used to simulate binding of
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27
N5-Pan and N7-Pan, placing the alkyl chains within a hydrophobic pocket containing
multiple aromatic residues. The two pantothenamides are substrates of EcPanK with Km
values of 140 and 128μM, respectively (Ivey, Zhang et al. 2004).
The PanK from Mycobacterium tuberculosis (MtPanK) is another type I enzyme
and shares approximately 52% sequence identity with EcPanK. Unlike EcPanK which
has a clear preference for ATP, MtPanK can use either ATP or GTP as phosphate donors
with equivalent efficiency. The structures of MtPanK in complex with multiple substrate
and product combinations lend a unique opportunity for structural comparisons with
EcPanK (Chetnani, Das et al. 2009; Chetnani, Kumar et al. 2010; Chetnani, Kumar et al.
2011). The structural properties of the active site of MtPanK are distinct from those of
EcPanK. While the EcPanK active site conformation has flexibility to accommodate
substrates and products, the MtPanK active site conformation is rigid and requires
significant substrate movements for product formation. Similar to EcPanK, MtPanK can
phosphorylate pantothenamides such as N-nonylpantothenamide (N9-Pan) (Chetnani,
Kumar et al. 2011).
1.6.2 Type II Pantothenate Kinases
Type II PanKs are found primarily in eukaryotic species. The first type II enzyme
characterized is the PanK from Aspergillus nidulans (AnPanK) that also has sequence
resemblance to the PanK of S. cerevisiae (Calder, Williams et al. 1999). However,
AnPanK has very low sequence homology with the well-characterized EcPanK.
Furthermore, whereas CoA is the strongest inhibitor of EcPanK, AnPanK is more
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28
strongly inhibited by acetyl-CoA (Calder, Williams et al. 1999). Differences in amino
acid sequence and regulatory properties between AnPanK and EcPanK have justified a
separate classification for AnPanK.
The first mammalian PanK discovered and characterized is the PanK from M.
musculus (mPanK) that has high sequence homology to AnPanK, but also bares little
resemblance to EcPanK (Rock, Calder et al. 2000). The mPanK1 gene encodes for two
alternatively spliced gene products; mPanK1α is expressed in the heart and kidney, and
mPanK1β is found in the liver and kidney. Like AnPanK, acetyl-CoA inhibits both
isoforms of mPanK more strongly than CoA with an IC50 of approximately 20μM (Rock,
Calder et al. 2000). However, CoA shows stimulatory activity that appears to be unique
to mPanK1β (Rock, Calder et al. 2000). Malonyl-CoA strongly inhibits the α-isoform,
but moderately so for the β-isoform (Rock, Karim et al. 2002). It is possible that
differential expression of mPanK1α and mPanK1β serves to regulate free CoA:esterified
CoA levels (Rock, Karim et al. 2002).
There are four subtypes of human PanKs (PANK1, PANK2, PANK3 and
PANK4) that were discovered when PANK2 was mapped out in connection with
pantothenate kinase-associated neurodegeneration (PKAN) (Zhou, Westaway et al.
2001). All four human PANK isoforms share a conserved catalytic core, and are
products of the differentially spliced PANK gene (Hong, Senisterra et al. 2007). PANK1
(containing isoforms α and β) is expressed in multiple organs including the heart, kidney
and liver. PANK2 is exclusively found in the brain (specifically, basal ganglia). PANK3
is expressed primarily in the liver (Zhou, Westaway et al. 2001). PANK4 is found
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29
mainly in muscle and has sequence similarity with S. cerevisiae and C. elegans (Zhou,
Westaway et al. 2001). As it lacks the essential glutamate residue required for kinase
activity, PANK4 is the only inactive isoform and its function is unknown (Hong,
Senisterra et al. 2007). The involvement of PANK2 mutations in neurodegeneration is
unclear though they are correlated with abnormal iron accumulation in the brain (Leoni,
Strittmatter et al. 2012).
Although type II enzymes are widely known to constitute the group to which
eukaryotic PanKs belong, some bacterial PanKs are classified into this class. Most
notable are the PanKs from staphylococci (S. aureus, S. epidermidis and S. haemolyticus)
as well as bacilli (B. cereus and B. subtilis) (Choudhry, Mandichak et al. 2003).
Phylogenetic analysis shows that SaPanK is a distant relative of the PanK from
Drosophila melanogaster (Choudhry, Mandichak et al. 2003). Unlike all previously
discovered type I and II PanKs, CoA and its thioesters do not inhibit SaPanK (Leonardi,
Chohnan et al. 2005). The lack of feedback regulation would lead to elevated levels of
CoA; S. aureus lacks glutathione and likely relies on CoA, a component of the CoA/CoA
disulfide reductase redox (CoADR) system, to relieve oxidative stress (Leonardi,
Chohnan et al. 2005).
The structure of SaPanK in complex with the ATP analogue AMPPNP is
available (Fig. 9B) (PDB: 2EWS) (Hong, Yun et al. 2006). Each subunit of the SaPanK
dimer is made up of actin-like domains that place the enzyme within the acetate/sugar
kinase/heat shock protein 70/actin (ASKHA) superfamily (Hurley 1996; Hong, Yun et al.
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30
2006). A Mg2+
ion coordinates the AMPPNP β and γ phosphates, which also interact
with the P loop and “pseudo-P loop” motifs of the actin domains.
The crystal structures of the human PANK1α and PANK3 in complex with
acetyl-CoA have been solved (PDB: 2I7N and 2I7P) (Hong, Senisterra et al. 2007).
Human PANK1α and PANK3 show a high affinity for acetyl-CoA, as extensive dialysis
and incubation with the ATP analogue AMPPNP can not dislodge the feedback regulator
from the active site (Hong, Senisterra et al. 2007). Like SaPanK, human PanK also
contains actin-like domains that resemble motifs of ASKHA family members. The
binding site of the pantetheine group of acetyl-CoA is located at the dimer interface; this
is in contrast to type I PanKs that do not share subunits for inhibitor/substrate binding
(Hong et al., 2007). The human structures provide the structural basis for stronger
inhibition of CoA thioesters versus CoA; the carbonyl group from the acetyl group forms
a hydrogen bond with a valine main chain amide nitrogen. Mutagenesis studies involving
thermostability assays, in conjunction with structural analysis of the two solved human
PanK isoforms, led to classification of PANK2 mutations (in connection with PKAN)
into three categories; mutations are either located at the dimer interface (affecting ability
to dimerize), the active site (affecting catalytic activity and/or capacity to bind
substrates), or the protein surface (to negatively affect thermostability) (Hong, Senisterra
et al. 2007).
No kinetic studies have been published regarding human PANKs using
pantothenamides as substrates. However, one study found that the pantothenamides N7-
Pan and N9-Pan showed potent IC50 values of 64 and 128μ/mL respectively when tested
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31
in human HepG2 liver cells (Choudhry, Mandichak et al. 2003). In addition, the structure
of human PANK3 in complex with N7-Pan (PDB: 3SMS) shows that the compound
occupies the pantothenate-binding site of the human enzyme.
1.6.3 Type III Pantothenate Kinases
Type III PanKs (also called coaX) are a recently discovered class with low
sequence homology to types I and II PanKs, and also show considerably different
structural and kinetic properties. Compared to type I PanKs, this third type has an even
wider distribution in the bacterial kingdom (Yang, Eyobo et al. 2006). Despite sharing
minimal similarity in sequence to types I and II PanKs, the remaining four enzymes of
the five-step CoA synthesis pathway are conserved (Brand and Strauss 2005). Km values
of type III PanKs for pantothenate are comparable to those of types I and II PanKs,
though Km values for ATP are unusually high in the millimolar range (Brand and Strauss
2005; Hong, Yun et al. 2006; Yang, Eyobo et al. 2006). Some bacteria such as
Mycobacteria express types I and III PanKs, though the latter is non-essential (Awasthy,
Ambady et al. 2010). In addition, unlike prokaryotic type I and eukaryotic type II
enzymes, type III PanKs are not feedback-regulated by CoA or its thioesters. Similar to
S. aureus, the lack of feedback regulation in bacilli (such as B. anthracis and B. subtilis)
can be justified also by a lack of glutathione and dependence on the CoADR redox
system for detoxification of oxidative stress (Nicely, Parsonage et al. 2007). Another
distinct feature of type III PanKs is the requirement of a monovalent cation, such as
NH4+, or K
+, for activity (Hong, Yun et al. 2006).
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32
The structures of the type III PanKs from Pseudomonas aeruginosa (PaPanK)
(Fig. 9C) (PDB: 2F9T) (Hong, Yun et al. 2006) and Thermotoga maritima (TmPanK)
(PDB: 3BEX) (Yang, Eyobo et al. 2006) are available. These enzymes contain actin-like
folds, like SaPanK, placing them in the ASKHA superfamily. However, they cannot use
pantothenamides as substrates. The PaPanK-pantothenate binary complex provides the
structural basis for resistance of type III-expressing bacteria to pantothenamides; the
portion of the substrate-binding site that interacts with the pantothenate carboxyl end
does not have additional space to fit any N-substitutions on pantothenamides (Hong, Yun
et al. 2006). The TmPanK-ADP-Pan ternary complex structure (PDB: 3BF1) (Yang,
Strauss et al. 2008) reveals the substrate-binding site to be at the dimerization interface;
like type II PanKs, the substrate is stabilized by binding to both subunits of the dimer.
Interestingly, some type III PanKs (such as those from P. aeruginosa and H.
pylori) have high sequence homology to the Bordetella pertussis Bvg accessory factor
(Baf) (Brand and Strauss 2005). Baf is a transcriptional regulatory protein that interacts
with the transcription factor Bvg to enhance the expression of the ADP-ribosylating
pertussis toxin (DeShazer, Wood et al. 1995; Wood and Friedman 2000; Williams,
Boucher et al. 2005).
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33
Figure 7. Phylogenetic distributions of prokaryotic and eukaryotic pantothenate
kinases from notable organisms. The phylogenetic tree shows the distribution of the
three types of PanKs. The human and murine PanKs are both of isoform 3, the subtype
containing only the conserved catalytic core. The tree was generated using the software
on www.phylogeny.fr, following alignment of sequences by ClustalW.
Type 1
Type 2
Type 3
http://www.phylogeny.fr/
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34
A.
-
35
B.
-
36
Figure 8. Sequence-based alignments of prokaryotic and eukaryotic PanKs from
types I (A), II (B), and III (C). Conserved (red) and similar (yellow) residues are
indicated.
C.
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37
Figure 9. Comparison of the structures and dimer folds of types I, II and III
bacterial PanKs. A. EcPanK (PDB: 1SQ5). B. SaPanK (PDB: 2EWS). C. PaPanK
(PDB: 2F9T). Each colour denotes a single subunit.
A.
C.
B.
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38
1.7 Hypothesis and Rationale for Study
Our working hypothesis is that structural characterization of the KpPanK
substrate-binding site will provide basis for design of specific KpPanK pantothenate
analogues to treat klebsiella infections.
KpPanK has high sequence homology with EcPanK (90.3% identity), which
contains an aromatic pocket towards the carboxyl end of the pantothenate substrate; it is
likely that KpPanK has a similar pocket that was proposed to accommodate
pantothenamide N-substitutions for EcPanK (Ivey, Zhang et al. 2004). This pocket
represents an empty space that can be occupied with N-substitutions for enhanced
binding affinity; we propose that chemical groups can be introduced to optimize
interactions with pocket residues. High sequence homology with EcPanK also suggests
that pantothenamides can also be phosphorylated as substrates by KpPanK. Next, a K.
pneumoniae contains a PanF similar to that found in E. coli. Moreover, the four
downstream enzymes involved in CoA biosynthesis are present in K. pneumoniae,
suggesting that these substrate analogues, as in E. coli, can lead to accumulation of CoA
derivatives, covalent inactivation of ACP and subsequent inhibition of fatty acid
synthesis.
1.7.1 Aims and Approaches
The primary objective of these studies is to solve the three-dimensional structure
of KpPanK by X-ray crystallography, an important technique that can allow us to
elucidate the architecture of its substrate-binding site at atomic resolution. Structural
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39
characteristics of the pantothenate-binding site can then be exploited for the design of
pantothenate derivatives that bind to KpPanK with high affinity.
1.7.2 Rationale for Experimiental Approach
1.7.2.1 Structure Determination of Macromolecules
Nuclear magnetic resonance (NMR) spectroscopy and X-ray crystallography
represent the two principal methods for structure determination of biological
macromolecules at the atomic level. Each method has its strengths and weaknesses.
NMR spectroscopy enables elucidation of atomic details of a protein in its solution state,
but is limited by the extensive amount of time required for solving one structure
(Gronwald and Kalbitzer 2010) as well as the size of the protein of interest; the technique
is ideally suited for proteins under 40 kDa in size (Doerr 2006). X-ray crystallography is
the method of choice for solving structures. This technique represents an efficient
method of macromolecular structure determination at atomic resolution without the
limitations of protein size and time restraints imposed by NMR (Feng, Pan et al. 2010).
To date, nearly 90% of over 80,000 structures deposited to the Protein Data Bank (PDB)
were solved by crystallography.
1.7.2.2 X-ray Crystallography
X-ray crystallography takes advantage of a protein crystal’s ability to scatter X-
ray beams (Bragg 1915). X-rays are diffracted by the electron cloud surrounding each
atom. Subsequently, diffraction patterns recorded by a detector can be used to recreate
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40
the electron density into which a model of the target protein can be built (Bragg 1915;
Rupp 2009).
1.7.2.3 Protein Crystallization
Protein overexpression and purification are necessary for providing a protein
sample of adequately high concentration and purity required for crystallization. A
common method for crystallization is vapour diffusion. Purified protein is first mixed
with a solution of precipitant (for example, ammonium sulfate or polyethylene glycol),
within a closed container over a large reservoir that holds the same solution. The
concentrations of both components are initially below that necessary to precipitate the
protein out of solution (Rhodes 2006). The water content of the mixture gradually
diffuses to the reservoir, thereby raising the concentrations of protein and precipitant to
cause precipitation; a crystal is the result of protein precipitated out of solution in an
ordered manner (Rhodes 2006). Crystal formation takes place in two stages: nucleation
and growth (Fig. 10A). First, protein molecules cluster together to “nucleate”, or to form
a seed. This is followed by the addition of protein molecules in solution to the seed
during crystal growth (Fig 10B).
Factors that can affect crystallization include pH, type of precipitant,
concentrations of protein and precipitant, protein purity and temperature (Rhodes 2006).
The use of screening kits is a practical method for determining an initial crystallization
condition. The fine-tuning of these factors may be necessary to produce optimally
diffracting protein crystals (this is commonly referred to as optimization).
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41
Figure 10. Phase diagram of protein crystallization. A. Sufficiently high
concentrations of protein and precipitant are necessary for nucleation and crystal growth
(blue); only crystal growth can be attained at lower concentrations (green). The red zone
indicates low concentrations that cannot support nucleation or growth. B. Large crystals
are ideally grown when peak protein and precipitant concentrations achieved are just
enough to achieve nucleation, followed by a shift to the green zone for crystal growth.
(from Crystallography Made Crystal Clear. Rhodes 2006, Figure 3.5).
A. B.
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42
1.7.2.4 Data Collection
Two key components of structure factors used to calculate an electron density
map are amplitude and phase (Rhodes 2006). The former can be acquired through data
collection, which involves obtaining information on the intensities of reflections (or
spots), the square roots of which are amplitudes of the structure factors. Data collection
consists of obtaining the diffraction patterns of the protein crystal in one-degree
increments; usually a total rotation of 180º (to obtain 180 frames) is sufficient to achieve
a complete data set.
The collection of x-ray diffraction data at extremely low temperatures (known as
cryocrystallography) such as in liquid nitrogen, is beneficial as it protects the crystal
against radiation damage (Rhodes 2006). A single crystal could then be used to collect a
complete data set, which otherwise would require several crystals if data collection took
place at room temperature. Ice crystals can form when protein crystals are frozen,
requiring the use of cryoprotectants (substances that prevent ice crystal formation).
1.7.2.5 Structure Determination
Following data collection, the first step in processing the data is indexing, which
involves finding the correct crystal symmetry space group based on the geometric
arrangement of reflections (ie. spots in a diffraction pattern) (Rupp 2009). In the
integration step, intensities are assigned to the reflections for all frames. Next, the
scaling of data merges all corresponding reflections between each frame into a single set
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43
of unique reflections (after removal of all outliers), and finds a consistent intensity scale
for all reflections.
Data collection can only provide information on structure factor amplitudes,
whereas phase information is lost. Molecular replacement (MR) is a common method
employed to solve this “phase problem” by using a similar structure as a search model.
MR searches for a correct solution by orienting the model such that it corresponds with
the observed amplitudes (Rossmann 1962; Evans and McCoy 2008). Next, the phases of
the model are “borrowed” and used to estimate phases of the unknown structure, which
are combined with experimentally determined amplitudes to calculate an electron density
map for the target protein.
2 MATERIALS AND METHODS
2.1 Materials
The KpPanK template gene (1-316) was synthesized by GenScript. The
expression vector pET28-MHL was developed in-house by the Structural Genomics
Consortium (Fig 11). Pfu UltraII DNA polymerase was purchased from Agilent
Technologies. Restriction enzymes for plasmid digestion were purchased from New
England Biolabs. Primers were synthesized by Eurofins Operon. PCR purification and
miniprep kits were purchased from Qiagen. Growth media (Luria-Bertani, and Terrific
Broth) were purchased from Sigma-Aldrich. Benzonase nuclease was purchased from
Novagen. DE52 anion exchange resin was purchased from Whatman. Nickel-
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44
nitrilotriacetic acid (Ni-NTA) resin beads were purchased from Qiagen. SDS-PAGE gels
(TGX 4-20%) were purchased from Biorad.
Adenosine diphosphate (ADP) was purchased from Sigma. The pantothenamides
used for structural and kinetic studies, N5-Pan, Np-Pan and compound 349, were
generously provided by our in-house chemists, Drs. David Smil and Yuri Bolshan. D-
pantothenic acid was purchased from Sigma-Aldrich. Crystallization screening kits were
developed and made in-house. 96-well plates (Art Robbins Intelliplates) used for
crystallization trials were purchased from Hampton Research. Proteases used for in situ
proteolytic treatment were purchased from Sigma. For crystal optimization, the Additive
Screen kit from Hampton Research was used.
For the kinase activity assay, the following were purchased from Sigma: pyruvate
kinase and lactate dehydrogenase enzymes, adenosine triphosphate (ATP),
phosphoenolpyruvate (PEP), and reduced β-nicotinamide adenine dinucleotide (NADH).
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45
Figure 11. Overview of the pET28-MHL expression vector. The vector encodes a
kanamycin resistance marker and a hexahistidine tag located N-terminal to the gene of
interest. The vector is first linearized by restriction enzyme digestion (at sites flanking
the SacB gene), and the SacB gene is replaced with the gene of interest upon ligation. A
powerful promoter, the T7 promoter, mediates rapid transcription of the inserted gene by
the T7 polymerase.
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46
2.2 Methods
2.2.1 Preparation of Expression Plasmid
KpPanK constructs were designed based on previously solved structures in the
PDB (specifically, EcPanK and MtPanK) as well as secondary structure predictions.
Gene inserts for each construct were amplified by polymerase chain reaction; primers
corresponding to each construct contain sequences that are complementary to BseRI
restriction enzyme recognition sites (Table I). A small amount of PCR products was
analyzed by electrophoresis on a 1% agarose gel to confirm the presence and size of
amplified gene inserts. For ligation of gene to vector, 1μL of PCR product was mixed
with 2µL of Infusion HD EcoDry pellet (Clontech) dissolved in linearized pET28-MHL
vector (pre-digested with BseRI enzyme). The mixture was incubated at 37ºC for 20
minutes, room temperature for 10 minutes and put on ice. The ligated mixture was then
transformed to E. coli DH5α cells, and plated onto LB agar plates (containing
kanamycin) and incubated overnight at 37ºC. Colonies confirmed with a positive gene
insert were chosen for growth, and the plasmid DNA was extracted by miniprep (Qiagen
Miniprep kit).
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47
Figure 12. Small scale test of expression of KpPanK constructs. The soluble portion
(left) and whole cell lysates (right) for each construct are shown. The arrow indicates the
protein bands corresponding to solubly-expressed KpPanK. The sizes of the standard
protein ladder markers on the left are indicated.
K1 K2 K3 K4 K5 K6 K7 10 kD
15 kD
20 kD
25 kD
37 kD
50 kD
75 kD
100 kD
150 kD
250 kD
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48
Table I. Sequences of primers used to generate KpPanK constructs of variable
truncation. The forward (ttgtatttccagggc) and reverse (caagcttcgtcatca) tail additions
correspond to the BseRI recognition sites. The start and end positions of each construct
are also indicated.
315 12 caagcttcgtcatcaACGTAAGCGTACTTGATTCACAG ttgtatttccagggcTACCTACAATTTAACCGCCACC K7
316 12 caagcttcgtcatcaTTTACGTAAGCGTACTTGATTCAC ttgtatttccagggcTACCTACAATTTAACCGCCACC K6
315 9 caagcttcgtcatcaACGTAAGCGTACTTGATTCACAG ttgtatttccagggcATGACACCGTACCTACAATTTAAC K5
316 9 caagcttcgtcatcaTTTACGTAAGCGTACTTGATTCAC ttgtatttccagggcATGACACCGTACCTACAATTTAAC K4
315 6 caagcttcgtcatcaACGTAAGCGTACTTGATTCACAG ttgtatttccagggcCAGACGTTAATGACACCGTAC K3
316 6 caagcttcgtcatca TTTACGTAAGCGTACTTGATTCAC ttgtatttccagggcCAGACGTTAATGACACCGTAC K2
316 1 caagcttcgtcatca TTTACGTAAGCGTACTTGATTCAC ttgtatttccagggcATGAGCCAAAAAGAGCAGACG K1
End Start Reverse Primer Forward Primer Construct
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49
2.2.2 Protein Expression and Purification
KpPanK plasmids were transformed into E. coli BL21(DE3) competent cells
(BL21 refers to a strain deficient in lon and ompT proteases, and DE3 designates an
IPTG-inducible T7 polymerase – explained below) by heat shock, and plated onto LB
agar and incubated at 37ºC overnight. The next day, Luria-Bertani (LB) broth (Sigma)
was inoculated with transformants and grown at 37ºC for 16 hours. Next morning, the
overnight LB broth culture was transferred into Terrific Broth (TB) (Sigma) and further
grown at 37 ºC to achieve an OD600 of ~0.7 before overnight induction with 1mM
isopropyl β-D-thiogalactopyranoside (IPTG) at 18 ºC for 16 hours (T7 polymerase
expression in BL21(DE3) cells is under the control of the lac operon, whereby the
allolactose analogue IPTG binds to and inactivates the lac repressor to induce T7
expression, leading to excess gene transcription). The cells were harvested next morning
by centrifugation, flash frozen with liquid nitrogen and stored at -80 ºC until purification.
Prior to the start of protein purification, cell pellet was thawed and resuspended in
buffer A (50mM Tris-HCl pH 8.0, 5% [v/v] glycerol, 300mM NaCl), and supplemented
with 5mM imidazole, 0.5% 3-[(3-cholamidopropyl)dimethylammonio]-1-
propanesulfonate, 5 units/mL benzonase, 1mM phenylmethylsulfonyl fluoride and 1mM
benzamidine. The cells were lysed by sonication using a Misonix Sonicator 3000 (10s
ON, 10s OFF, for a total of 20 minutes at power output ~120W). The lysate was then
clarified by centrifugation (16000rpm for 90 minutes using a Beckman Coulter J-20 XPI
Centrifuge fitted with a JLA 16.250 rotor) and the supernatant was loaded into an open
column (Biorad Econo) containing DE52 resin (pre-charged with 2.5M NaCl) (DE52 is
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50
an ionic exchange resin that is used both to capture anionic molecules such as nucleic
acids, and to filter the lysate). The flow-through from the first column drips onto a
second open column containing nickel nitrilotriacetic (Ni-NTA) resin beads to which
hexahistidine tagged proteins bind with high affinity. Once the lysate had passed
through, the Ni-NTA beads were washed with 50mL buffer A containing 30mM
imidazole. The protein was then eluted using 10mL of buffer A containing 500mM
imidazole (Fig. 13A).
The protein sample was further purified by size exclusion chromatography (SEC)
using Superdex 75 resin that was pre-equilibrated with gel filtration buffer (20mM Tris-
HCl pH 8.0, 5% glycerol, 200mM NaCl). The purity of each fraction was assessed using
SDS-PAGE gels (Fig. 13B); fractions of the highest purity were pooled together. The
molecular weight of the protein was verified by mass spectrometry.
Types I and II PanKs are known to bind with high affinity to CoA and its
thioesters, which show up in the crystal structures despite extensive dialysis (Hong,
Senisterra et al. 2007; Chetnani, Das et al. 2009). To remove co-purified substrates or
inhibitors, the protein was dialyzed for 3 days in 20mM Tris-HCl pH 8.0. The protein
was then concentrated to 35mg/mL using centrifugal filter units (Amicon 15mL size with
10kDa cutoff). Protein concentration was verified by triplicate measurements using the
NanoDrop 1000 Spectrophotometer (this instrument measures UV absorbance at 280nm,
which is due mainly to tryptophan and tyrosine residues).
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51
Figure 13. Purification of KpPanK. A). SDS-PAGE gel of the wash flowthrough (left)
and eluted protein (right) during Ni-NTA affinity purification. (Note: The protein was
purified by splitting the sample into two open columns.) B). SDS-PAGE gel of gel
filtration peak fractions.
HiLoad 26 60 S75001:10_UV HiLoad 26 60 S75001:10_Fractions
0
500
1000
1500
2000
mAU
120 140 160 180 200 220 ml
A1 A2 A3 A4 A5 A6 A7 A8 A9 A10 A11 A12 B12 B11 B10 B9 B8 B7 B6 B5 B4 B3 B2 B1
A10 A11 B12 B11 B10 A12 10 kD
15 kD
20 kD
25 kD
37 kD
50 kD
75 kD
100 kD
150 kD
250 kD B.
10 kD
15 kD 20 kD 25 kD 37 kD
50 kD
75 kD 100 kD
150 kD
250 kD
Wash Elution
A.
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52
2.2.3 Protein Crystallization and Data Collection
KpPanK protein was incubated with 5mM MgCl2, 30mM ADP and 30mM
substrate (pantothenate, N5-Pan, N7-Pan, Np-Pan or compound 349) overnight at 4ºC.
The inclusion of ADP is due to sequential substrate binding observed in the type I E. coli
PanK (ie. nucleotide binds first, followed by substrate) (Song and Jackowski 1994). The
diphosphate form was chosen over the triphosphate form to prevent formation of a
phosphorylated product which can be released easily. The protein was then mixed at 1:1
(0.5μL) ratio with solutions from two in-house screening kits (containing 96 conditions
each) using a Rigaku Phoenix-HT liquid-handling robot, and crystallized using the sitting
drop vapour diffusion method in 96-well Intelliplates. In situ proteolysis was also used to
increase the success rate of crystallization (Dong, Xu et al. 2007). Briefly, this method
involves the addition of trace amounts of protease to the buffer:protein mixture for the
purpose of truncating flexible polypeptides to yield more globularly shaped proteins
(favourable for crystallization). In these studies, 1:500 ratio by weight of protease to
protein was added (e.g. 1mg of protease per 500 mg of protein). The proteases used
include: α-chymotrypsin, trypsin, elastase, subtilisin, endoproteinase Glu-C V8, papaya
proteinase I, dispase I and thermolysin.
Within a week, crystals appeared in a condition containing 20% (w/v) PEG3350
and 0.2M tri-lithium citrate in drops that did not contain proteases (Fig 14A, 15A).
Though crystals also appeared when proteases were supplemented, initial attempts at
optimization omitted proteases. Crystals were transferred to a cryoprotectant solution
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53
containing 1:1 mixture of paratone-N and mineral oil, and stored in liquid nitrogen for
screening/data collection.
Dr. Wolfram Tempel (SGC, Toronto) generously provided technicial assistance
in screening crystals using the in-house X-ray generator (Rigaku Rotating Copper Anode)
in the Structural Genomics Consortium (SGC). Among crystals from the original
condition and several initial rounds of optimization (altering PEG3350 and tri-lithium
citrate concentrations), the best resolution was 3.5Å. The diffraction quality of the
crystals was significantly improved by growing them in the mixture mentioned
previously and supplementing 0.2 μL of additives from the Hampton Research Additive
Screen kit (96 additives): (±)-1,3-butanediol helped improve N5-Pan bound crystal
diffraction to 2.1Å (Fig. 14B); 2,5-hexanediol improved the resolution of crystals from
protein incubated with Np-Pan (Fig. 15B).
Data used to solve the final structures were collected at the Advanced Photon
Source (Argonne National Laboratory, IL, USA). Diffraction data for KpPanK
complexed with N5-Pan were collected using 19-ID beamline. Data for KpPanK
complexed with Np-Pan were collected using the 23-IDB beamline. Dr. Wolfram
Tempel and ANL staff generously provided assistance in collecting X-ray diffraction
data.
Diffraction data were indexed and integrated by using the program XDS (Kabsch
2010), and processed and scaled by Pointless and Scala (Evans 2006) in the CCP4 suite
(Collaborative Computational Project 1994).
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Figure 14. Crystals of KpPanK co-crystallized with N5-Pan. A. Initial crystals of
N5-Pan bound KpPanK, grown by mixing 0.5μL protein (35mg/mL incubated with
30mM N5-Pan, 30mM ADP and 5mM MgCl2) and 0.5μL reservoir buffer (20% w/v
PEG3350, 0.2M tri-lithium citrate). These crystals diffracted with an average resolution
of 3.5Å. B. Optimized crystals of KpPanK incubated with N5-Pan grown by adding
0.2μL 40% (±)-1,3-butanediol to the mixture mentioned. The best crystal diffracted to
2.1Å resolution.
B.
A.
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55
Figure 15. Crystals of KpPanK co-crystallized with Np-Pan. A. Initial crystals of
Np-Pan bound KpPanK, grown by mixing 0.5μL protein (35mg/mL incubated with
30mM Np-Pan, 30mM ADP and 5mM MgCl2) and 0.5μL reservoir buffer (20% w/v
PEG3350, 0.2M tri-lithium citrate). B. Optimized crystals of Np-Pan bound KpPanK
grown by adding 0.2μL 2,5-hexanediol to the mixture mentioned. The best crystal
diffracted to 1.95Å resolution.
A.
B.
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56
Table II. Summary of substrates used for KpPanK co-crystallization and the best
resolution achieved.
1.95 Å yes N-pyridin-3-
ylmethylpantothenamide
(Np-Pan)
4.0Å yes Compound 349
>10.0Å yes N-heptylpantothenamide
(N7-Pan)
2.1Å yes N-pentylpantothenamide
(N5-Pan)
>10.0Å yes Pantothenate
Best Resolution Successful crystallization
Chemical
Structure
Compound
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57
2.2.4 Structure Determination, Refinement and Validation
The structure of KpPanK complexed with N5-Pan was solved by molecular
replacement using the program PHASER-MR (Read 2001) and one monomer of the E.
coli PanK (PDB: 1SQ5) as a search model (EcPanK is a suitable model because it shares
90% sequence identity with KpPanK). The output from XDS following indexing and
integration provided the unit cell dimensions of the KpPanK crystals, and indicated the
lattice with the highest symmetry space group to be primitive orthorhombic (P222) (a
crystal lattice in the form of a rectangular prism defined by 90º crystallographic axes).
The number of molecules in the asymmetric unit could be predicted using the Matthews
coefficient (volume of the unit cell divided by the product of the protein’s molecular
weight, number of asymmetric units per unit cell and the number of molecules per
asymmetric unit). Matthews probability calculation (Matthews 1968; Kantardjieff and
Rupp 2003) indicated that there are most likely eight or nine monomers per asymmetric
unit (Fig. 17), based on empirically observed Matthews coefficients from structures
deposited in the PDB. However, since the known functional unit of type I PanKs is a
homodimer (Song and Jackowski 1994), eight subunits were entered as a search
parameter in molecular replacement. The high resolution limit was set to 4Å in
PHASER-MR to allow for possible conformational differences in the side chains between
the KpPanK structure and the search model. The space groups belonging to the primitive
orthorhombic class (P222, P2221, P21212 and P212121) were tested to find the correct
molecular replacement solution. Of the tested space groups, the group P212121 showed
the best solution (ie. no clashes were observed between the subunits when the model was
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58
displayed on screen). After one round of model refinement, the working and free R
factors were 28% and 32%, respectively.
Manual model building was done using the program Coot (Emsley and Cowtan
2004; Emsley, Lohkamp et al. 2010). Water molecules were built in Coot with the
following restrictions with the following requirements: visible density with hydrogen
bond distances between 2.2 and 3.2Å. Uninterpretable density was modeled as unknown
(UNX) atoms. The model was refined using Refmac5 (Murshudov, Skubak et al. 2011)
in the CCP4 suite. Hydrogen atoms were generated for refinement in Refmac5, but were
excluded in the coordinate output.
For KpPanK in complex with Np-Pan, the diffraction data were integrated,
indexed and scaled as with the N5-Pan structure. Indexing by XDS also indicated nearly
identical unit cell dimensions and space group as the N5-Pan structure. As such, the
coordinates of the KpPanK·N5-Pan structure were used to refine against the newly scaled
data of the KpPanK·Np-Pan complex after removing all water molecules and bound N5-
Pan. The working and free R factors from an initial round of refinement were 24% and
28% respectively. Water molecules were added using Coot, as mentioned in model
building of the KpPanK·N5-Pan complex.
The restraints for the chemical structures of ligands (N5-Pan and Np-Pan) used in
refinement were generated using the PRODRG server (Schuttelkopf and van Aalten
2004). The Molprobity server was used to validate both structures for bond angle and
length variations, Ramachandran outliers and proper amino acid rotamers (or
conformation of side chains) (Chen, Arendall et al. 2011). All figures were generated
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59
using PyMOL (The PyMOL Molecular Graphics System, Version 1.5.0.3, Schrödinger,
LLC).
Statistics for data collection and refinement are shown in Table III. The KpPanK
structures in complex with N5-Pan and Np-Pan have been deposited to the PDB with
accession codes 4F7W and 4GI7, respectively.
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60
Figure 16. Diffraction patterns of KpPanK crystals. A. Diffraction pattern of a
crystal when screened with the SGC in-house X-ray generator Rigaku (model FR-E)
containing a rotating copper anode. B. Diffraction pattern of obtained during data
collection using the 19-ID beamline (Advanced Photon Source, Argonne National
Laboratory, IL).
A.
B.
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61
Ligand N5-Pan Np-Pan
PDB ID 4F7W 4GI7
Data Collection
Beamline 19-ID Advanced Photon Source
(APS)
23-IDB (APS)
Wavelength (Å) 0.97931 1.03321
Resolution (Å) 48.04-2.05 40-1.95
Space Group P212121 P212121
No. of molecules in asymmetric
unit
8 8
Unit Cell Parameters (Å) a = 127.8, b = 130.8,
c = 190.2
a = 127.9, b = 130.9,
c = 193.0
(degrees) α = β = γ = 90 α = β = γ = 90
No. of measured reflectionsa
1327648 (181294) 1668077 (214666)
No. of unique reflections 187519(27124) 234904 (33760)
Completeness (%) 99.7(99.3) 99.9 (99.1)
Friedel Redundancy 7.1(6.7) 7.1 (6.4)
/σ 13.7(3.0) 9.3 (2.1)
Rmergeb (%) 14.8(89.8) 12.9(85.2)
Refinement
Resolution (Å) 40.0-2.1 40-2.0
Rwork/Rfree (%)c
18.7/22.7 22.2/25.7
No. of atoms
protein 19392 19896
ligand/ion 389 372
water 988 1023
Average B-factors (Å2)
protein 26.3 33.4
ligand/ion 24.4 32.8
water 25.9 32.8
RMSD bond length (Å) 0.013 0.010
RMSD bond angle (degrees) 1.4 1.4
Ramachandran Analysisd
Favored (%) 91.6 91.9
Additionally allowed (%) 8 7.7
Generously allowed (%) 0.2 0.4
Disallowed (%) None None
Table III. Data collection and refinement statistics for KpPanK crystals.
a Numbers in parentheses are for the outer shell.
b Rmerge = Σ[(I − I )]/Σ(I), where I is the observed intensity and is the average intensity.
c Rwork = Σ[|Fobs| − |Fcalc|]/Σ|Fobs|, where |Fobs| and |Fcalc| are magnitudes of observed and calculated
structure factors respectively. Rfree was calculated as Rwork using 5.0% of the data, which was set
aside for an unbiased test of the progress of refinement.
d The program PROCHECK(Laskowski 1993) was used.
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62
Figure 17. Matthews Probability calculation of the oligomeric state of the KpPanK
asymmetric unit. The probabilities for the oligomeric state of the KpPanK asymmetric
unit were calculated based on the crystal’s diffracting resolution, unit cell dimensions,
space group, as well as the protein’s molecular weight. The highest probable oligomeric
states are located near the top of each peak.
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63
2.2.5 Spectrophotometric assessment of substrate kinetics
KpPanK substrate kinetics were examined based on an adapted protocol of the
pyruvate kinase/lactate dehydrogenase (PK/LDH) coupled assay publ