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) 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. ii 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 iii 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. iv 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’- 16-17 phosphopantetheine 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 v 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 vi 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 56 best resolution achieved. 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 82 substrates in KpPanK, EcPanK and MtPanK structures. vii 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 33 pantothenate kinases from notable organisms. Figure 8 Sequence-based alignments of prokaryotic and eukaryotic PanKs 34-36 from types I (A), II (B), and III (C). Figure 9 Comparison of the structures and dimer folds of types I, II and III 37 bacterial PanKs. 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 62 KpPanK asymmetric unit. Figure 18 Pyruvate kinase (PK)/lactate dehydrogenase (LDH) coupled assay 64 for characterization of kinase activity. Figure 19 Structure of a KpPanK subunit. 67 viii 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 73 residues. Figure 23 Michaelis-Menten plot of reaction velocity vs. substrate 75 concentration. Figure 24 Structural differences between KpPanK and EcPanK substrate 79 binding sites. 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- 86 binding site. Figure 27 Modeling of a branched derivative of Np-Pan in human PanK3. 87 ix 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 x 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 xi 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).