STRUCTURAL INVESTIGATIONS OF SELECTED Ph.D. Thesis NISHANT KUMAR VARSHNEY VARSHNEY KUMAR NISHANT Thesis Ph.D. MICROBIAL PENICILLIN G ACYLASES AND A PROTOZOAL N- MYRISTOYLTRANSFERASE Thesis Submitted to University of Pune For the degree of DOCTOR OF PHILOSOPHY In BIOTECHNOLOGY By NISHANT KUMAR VARSHNEY Under the guidance of Dr. C. G. SURESH DIVISION OF BIOCHEMICAL SCIENCES MAY CSIR-NATIONAL CHEMICAL LABORATORY PUNE – 411008, INDIA 2 013 MAY 2013 “Structural investigations of selected microbial penicillin G acylases and a protozoal N- myristoyltransferase” Thesis Submitted to University of Pune For the degree of DOCTOR OF PHILOSOPHY In BIOTECHNOLOGY By NISHANT KUMAR VARSHNEY Under the guidance of Dr. C. G. SURESH DIVISION OF BIOCHEMICAL SCIENCES CSIR-NATIONAL CHEMICAL LABORATORY PUNE – 411008, INDIA MAY 2013 Dedicated to…….. My Mom, Abha, Pranshi & the ALMIGHTY CERTIFICATE Certified that the work incorporated in this thesis entitled, “Structural investigations of selected microbial penicillin G acylases and a protozoal N-myristoyltransferase” submitted by Mr. Nishant Kumar Varshney was carried out by the candidate under my supervision. The data obtained from other sources has been duly acknowledged in the thesis. Date: (Dr. C. G. Suresh) Pune Research Guide DECLARATION OF THE CANDIDATE I hereby declare that the thesis entitled “Structural investigations of selected microbial penicillin G acylases and a protozoal N-myristoyltransferase” is entirely original and was carried out by me under the supervision of Dr. C. G. Suresh at the Division of Biochemical Sciences, National Chemical Laboratory (NCL), Pune, India. The thesis is submitted by me to the University of Pune for the degree of Doctor of Philosophy in Biotechnology. I further declares that the scientific contents of this thesis has not formed the basis for award of any degree, diploma, associateship, fellowship, titles in this or any other University or other institution of Higher learning. The material obtained from other sources has been duly acknowledged in the thesis. Nishant Kumar Varshney Date: Biochemical Sciences Division, CSIR-National Chemical Laboratory, Dr. Homi Bhabha Road, Pune- 411008. India CONTENTS Title Page Number Acknowledgements i List of Abbreviations ii Abstract vii Chapter 1: Introduction and Review of Literature 1-63 1.1. Introduction to Enzymes 1 1.2. β-lactam Antibiotics 3 1.3. History of β-lactam Antibiotics 3 1.4. Bacterial Resistance and Need for Semi-synthetic Antibiotics 6 1.5. Semi-synthetic Penicillins 7 1.6. β-lactam acylases 10 1.7. β-lactam acylases in the Production of Semi-synthetic Penicillins 11 1.8. Cephalosporin acylases 13 1.9. Penicillin acylases 16 1.9.1. Penicillin V acylases (type I) 16 1.9.2. Penicillin G acylases (Type II) 19 1.9.3. Ampicillin acylase (Type III) 19 1.10. Industrial Applications of Penicillin acylases 22 1.11. Biological Function of Penicillin acylases 22 1.12. Ntn-hydrolase Superfamily 23 1.13. Members of the Ntn-hydrolase Family 24 1.14. Post-translational Activation of Ntn-hydrolases 32 1.15. Autocatalytic Processing of PGAs 33 1.16. Structural Studies of PGA from E.coli 34 1.17. Catalytic Mechanism of PGAs 36 1.18. Studies on PGAs from other Sources 40 1.19. Important Residues for Catalysis and Autoproteolysis of PGAs 42 1.20. Industrial Applications of PGAs 44 1.20.1. 6-APA Production 44 1.20.2. Ring Expansion 45 1.20.3. Industrial Production of β-Lactam Antibiotics 45 1.20.4. Penicillin Acylase-catalyzed Peptide Synthesis 46 1.20.5. Penicillin acylase-catalyzed Resolution of Racemic Mixtures 46 1.21. Post-translational Modification and Myristoylation 47 1.22. Myristoyl-CoA:protein N-myristoyltransferase (NMT) 48 1.23. In vivo role of N-terminal Myristoylation of proteins 49 1.24. Structural Studies on NMTs 50 1.25. Catalytic Mechanism of N-Myristoylation 53 1.26. NMT as a Potential Drug Target 54 1.26.1. NMT as Antifungal Target 55 1.26.2. NMT as Antiviral Target 56 1.26.3. NMT as Anticancer Target 58 1.26.4. NMT as Antiparasite Target 59 1.27. NMT in Leishmaniases 60 1.28. Scope of the Thesis 62 Title Page Number Chapter 2: Materials and Methods 65-111 2.1. Introduction 65 2.2. Materials and Instruments used in the Study 67 2.3. Methods 2.3.1. Isolation, Cloning, Expression and Purification 69 of “pac” gene from Kluyvera citrophila DMSZ 2660 (KcPAC) 2.3.2. Cloning, Expression and Purification of N-terminal 76 deletion Mutant of NMT gene from Leishmania donovani (∆N LdNMT) 2.3.3. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 81 2.3.4. Staining using Coomassie blue 81 2.3.5. Silver Staining of PAGE gel 81 2.3.6. Biochemical and Biophysical Techniques for Protein 82 Characterization 2.3.7. Protein Preparation for Crystallographic Studies 89 2.3.8. Crystallographic Methods 90 2.3.9. Calculation of Thermostability Factors 111 Chapter 3: Cloning and Over-expression of pac gene from Kluyvera 112-139 citrophila and Purification and Biochemical Characterization of the Expressed KcPGA 3.1. Introduction 112 3.2. Results 3.2.1. Isolation and Cloning of “pac” gene from Kluyvera 115 citrophila DMSZ 2660 (KcPAC) 3.2.2. Amino Acid Sequence Analysis 122 3.2.3. Preparing Site-directed Mutants 122 3.2.3. Expression of KcPGA Proteins 123 3.2.5. Purification of KcPGA Proteins 124 3.2.6. Preliminary Biochemical Characterization of KcPGA 127 Wild-type Protein 3.3. Discussion 138 Chapter 4: Structural and folding studies of wild-type enzyme and slow- 140-171 processing mutant of KcPGA 4.1. Introduction 140 4.2. Results 142 4.2.1. Structural Studies of KcPGA unprocessed Precursor Mutant 142 4.2.2. Steady State Fluorescence Studies of Wild-type Processed 157 KcPGA 4.3. Discussion 169 Chapter 5: Structural studies of PGA from Alcaligenes faecalis 172-200 5.1. Introduction 172 Title Page Number 5.2. Results 175 5.2.1. Crystallization of AfPGA 175 5.2.2. Protein Preparation 176 5.2.3. Crystallization of AfPGA 176 5.2.4. Data Collection and Processing 177 5.2.5. Structure Determination 179 5.2.6. Model Building and Refinement of the Structure 180 5.2.7. Structure Validation 180 5.2.8. Description of the Structure 183 5.2.9. Calcium Site 189 5.2.10. Disulphide Bond 189 5.2.11. Structural Comparison of AfPGA Structure with other 191 PGAs 5.2.12. Thermostability Factors 194 5.3. Discussion 199 Chapter 6: Cloning, Over-expression, Purification and Crystal Structure 201-230 studies of N-myristoyltransferase from Leishmania donovani 6.1. Introduction 201 6.2. Results 204 6.2.1. Cloning of N-terminal Mutant of the LdNMT gene 204 6.2.2. Expression Studies for the Cloned Protein 206 6.2.3. Comparison of Expression and Initial Purification of 207 ∆N LdNMT in LB and in Auto Induction Medium (AIM) 6.2.4. Purification of ∆N LdNMT Enzyme 208 6.2.5. Crystallization of ∆N LdNMT 211 6.2.6. Soaking of the Crystals in the Reported Inhibitor of 212 L.major NMT and T.brucei NMT 6.2.7. Data Collection for the Soaked Crystals 213 6.2.8. Solvent Content of Crystals 216 6.2.9. Structure Solution 216 6.2.10. Refinement of the Structures 216 6.2.11. Structure Validation 219 6.2.12. Description of the Structure 220 6.2.13. Co-Crystallization of ∆N LdNMT with Substrate and 227 Inhibitor 6.3. Discussion 228 Chapter 7: Conclusions 231-234 Bibliography 235-276 Curriculum vitae 277-281 ACKNOWLEDGEMENTS The work that has been wrapped up inside the cover of this thesis could not have been possible without help, support and encouragement from a lot of people around me. I now take the opportunity to thank all who have helped me during these years though I might forget to mention a few of them. First and foremost, I would like to thank my mentor, Dr. C. G. Suresh, who has provided generous support and understanding throughout my stay at NCL. From him I have not only learned the working etiquettes and fundamentals of crystallography in great details but have also learned the art of doing things patiently and to perfection. He has given me all the freedom to think and plan my experiments independently however; whenever I was stuck he was always available with his advice and critical suggestions which I needed. I am very thankful to him for providing many opportunities for me to learn new things and to test my skills. I am indebted to him for his unflinching encouragements, freedom and cultivating good work culture in me. I would also like to thank my other supervisor, Prof. Anthony J. Wilkinson, for accepting me as a Split Site scholarship student in his lab in YSBL for an year during my PhD, helping me to grow in science, inducing my intellectual abilities as well as for being accepting of my eccentricities. Thanks for being very cooperative, warm, patient on my mistakes and failures and for his continuous monitoring and suggestions for best improvement of my research during and after the visit. I am grateful to Dr. James Brannigan for teaching me the principles of molecular biology and protein purification. I am very much grateful to him for his precious advice and constantly checking the progress of my research during my YSBL visit. I fall short of words to express my feelings towards Dodson family. I am deeply grateful to them for hosting me in their home during my stay in York, UK. Late Prof. Guy Dodson was always been inspirational and the valuable discussions in his living room besides the fire place have helped me tremendously in understanding the wonder science of crystallography.