Structural and Inhibition Studies on Udp-Galactopyranose Mutase
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STRUCTURAL AND INHIBITION STUDIES ON UDP-GALACTOPYRANOSE MUTASE A Thesis Submitted to the College of Graduate Studies and Research in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy In the Department of Chemistry University of Saskatchewan By Sarathy Karunan Partha © Copyright Sarathy Karunan Partha, October 2010. All rights reserved Permission to Use In presenting this thesis in partial fulfilment of the requirements for a Postgraduate degree from the University of Saskatchewan, I agree that the Libraries of this University may make it freely available for inspection. I further agree that permission for copying of this thesis in any manner, in whole or in part, for scholarly purposes may be granted by the professor or professors who supervised my thesis work or, in their absence, by the Head of the Department or the Dean of the College in which my thesis work was done. It is understood that any copying or publication or use of this thesis or parts thereof for financial gain shall not be allowed without my written permission. It is also understood that due recognition shall be given to me and to the University of Saskatchewan in any scholarly use which may be made of any material in my thesis. Requests for permission to copy or to make other use of material in this thesis in whole or part should be addressed to: Head of the Department of Chemistry University of Saskatchewan Saskatoon, Saskatchewan (S7N 5C9) i ABSTRACT UDP-galactopyranose mutase (UGM) is a flavoenzyme which catalyzes the interconversion of UDP-galactopyranose (UDP-Gal p) and UDP-galactofuranose (UDP- Gal f). UDP-Gal f is the active precursor of Gal f residues. Glycoconjugates of Gal f residues are found in the cell wall of bacteria and on the cell surface of higher eukaryotes. Gal f residues have not been found in humans and the fact that they are essential for the growth of pathogenic bacteria makes UGM a potential antibacterial target. In the present study, crystal structures of UGM from Deinocococcus radiodurans (drUGM) in complex with substrate (UDP-Gal p) were determined. UDP-Gal p is buried in the active site and bound in a U-shaped conformation. The binding mode and active site interactions of UDP-Gal p are consistent with the previous biochemical and mechanistic studies. The mobile loops in the substrate complex structures exist in a closed conformation and Arg198 on one of the mobile loops stabilizes the phosphate groups of the substrate. The anomeric carbon of galactose is 2.8 Å from the N5 of FAD (in the reduced complex) favorable to form FAD-galactosyl adduct. In addition to substrate complex structures, the crystal structures of drUGM in complex with UDP, UMP, and UDP-Glc have been determined. The mobile loops in all these complexes exist in a closed conformation. Inhibitors for UGM were identified by ligand-based and structure-based methods. The phosphonate analog of UDP-Gal p (GCP) showed only weak inhibition against various bacterial UGMs. The structure of drUGM in complex with GCP provided a basis for its inhibitory activity. Poor stabilization of the phosphate groups by conserved arginines (Arg198 and Arg305) and altered sugar binding mode account for its activity. ii Novel indole-based (LQ1, LQ6 and LQ10) inhibitors of UGM were identified through structure-based virtual screening (SBVS) of a chemical library. Inhibition studies also allowed the identification of an active site aspartic acid that plays role in inhibitor binding. The structural studies on drUGM provided a basis for understanding substrate binding to UGM. In vitro enzyme inhibition studies allowed the identification of novel indole-based inhibitors. The structural and inhibition studies reported here enhance the understanding of UGM-ligand interactions and will assist in the development of more potent inhibitors of UGM. iii ACKNOWLEDGEMENTS I feel deeply indebted to my supervisor, Dr. David Sanders, for giving me this opportunity of carrying out this exciting project in his laboratory. I am most thankful for his never-ending interest in the progress of this project and for teaching protein X-ray crystallography. The amount of knowledge and confidence I gained from him during these years is immense. I thank Dr. Sanders for showing huge interest to answer my questions seriously and for all his scientific and non-scientific advice throughout my PhD program. I thank Dr. Sanders, for providing me the opportunity to attend American Crystallographic Association 2009, Toronto to present some of the structural results of this dissertation. I would like to thank Dr. David Palmer for his invaluable suggestions for the project and answering my questions without any hesitation. It was a great pleasure and learning experience with Dr. David Palmer and I enjoyed the classes he taught to me. I would like to thank the present and past members of Dr. Sanders’ lab for making my stay at UofS enjoyable and memorable. I thank Dr. van Straaten for spending a lot of time in teaching me X-ray crystallographic techniques from data collection to structure solution. Special thanks to Josiah for helping me to learn many basic lab techniques in the initial stages of my program. I would like to thank the committee members, Dr. Grochulski, Dr. Pedras and Dr. Reid for their constructive criticism and valuable suggestions. I would like to thank Department of Chemistry for providing me the financial support over the period of my iv studies. Also, I would like to thank all the staff members (Virginia, Kathy, Dwight, Tanis, Devin, Garth, Ronda), Department of Chemistry for all their help and support. I would like to thank Dr. David Jakeman (Dalhousie University) for generously providing samples of UDP-Gal p analog for structural and inhibition studies. I express my sincere thanks to Dr. Todd Lowary (University of Alberta) for accepting me to visit his lab to gain technical expertise (UDP-Gal f synthesis). I would like to thank my parents Karunan and Sarojini who generously allowed me to pursue doctoral studies abroad. Their blessings and support is a “vitamin” for me to achieve this task. I am grateful to have my wife Viji for her tremendous love, support, motivation and understanding throughout this program. I would also like to thank my sisters (Geetha and Lakshmi) and brother (Bhaskar) for their support and encouragement. Finally, I am grateful to have friends like Visu, Venkat and Anand from my undergraduate studies back in India. I am unable to put in words about their support and encouragement over the period of my study. I would like to thank all the friends and families in Saskatoon for their support throughout the period of my study. My special thanks to Dr. Swarnam Ravindran and Vijay Mamillapalle for their support throughout the good and bad times. v DEDICATION I dedicate this dissertation to my PPParentsParents Sarojini and Karunan vi LIST OF TABLES Table 1-1 Kinetic parameters for kpUGM active site mutants…………………………..16 Table 2-1 Various methods for protein crystallization…………………………………..31 Table 3-1 PCR reaction components for site-directed mutagenesis…………………….46 Table 3-2 Reaction conditions for PCR…………………………………………………46 Table 4-1 Sequence numbers for conserved active site residues of UGM from K. pneumoniae , E. coli , M. tuberculosis and D. radiodurans …………………….62 Table 4-2 Kinetic data for drUGM and other bacterial UGMs………………………...64 Table 4-3 Data collection and refinement statistics for drUGM ox , drUGM red and drUGM:UDP complexes………………………………………………..65 Table 4-4 Distance between C α of Arg198 (drUGM ox ) and the corresponding arginine in other bacterial UGMs………………………………………………………76 Table 4-5 Data collection statistics and refinement for drUGM:UMP complex………..84 Table 4-6 Data collection and refinement statistics for drUGM:UDP-Glc complex……86 Table 5-1 Inhibitory activity of GCP towards kpUGM, mtUGM and drUGM………….95 Table 5-2 Data collection and refinement statistics for drUGM:GCP complex…………96 Table 5-3 Examples for successful application of structure-based virtual screening…..105 Table 5-4 Inhibitory profile towards kpUGM and mtUGM……………………………109 Table 5-5 Inhibition of kpUGM and mtUGM by LQ6 and LQ10 at 1 µM…………….109 Table 5-6 Inhibitory profile against drUGM WT………………………………………114 Table 5-7 Kinetic parameters for drUGM N372D ……………………………………..116 Table 5-8 Data collection statistics and refinement for drUGM N372D:UDP-Gal p complex………………………………………………………………………………....117 Table 5-9 Comparion of inhibitory profile between drUGM WT and drUGM N372D …………………………………………………………………….......120 vii Table 5-10 Inhibition of kpUGM WT and D351N mutant by LQ10………………….120 viii LIST OF FIGURES Figure 1-1 Cell wall architecture of Gram-positive and Gram-negative bacteria……….2 Figure 1-2 Schematic representation of the cell wall architecture of Mycobacterium to highlight the presence of Gal f residues…………………………....2 Figure 1-3 UGM catalyzed interconversion of UDP-Gal p and UDP-Gal f………….…..4 Figure 1-4 Biosynthesis of UDP-Gal f…………………………………………………...5 Figure 1-5 FAD structure and proposed S N1 or SET pathways for UGM mediated interconversion of UDP-Gal p. ……………………………………………………….10 Figure 1-6 Proposed S N2 pathway for UGM reaction………………………………….11 Figure 1-7 Structure of ecUGM………………………………………………………..13 Figure 1-8 Structure of reduced kpUGM………………………………………………14 Figure 1-9 Effect of substrate concentration on the initial velocity of an enzyme-catalyzed reaction……………………………………………………….…19 Figure 1-10 Schematic representation of the three types of reversible inhibition……………………………………………………………………………....22