STRUCTURAL AND FUNCTIONAL ASPECTS OF PYRANOSE-FURANOSE MUTASES By Jijin Raj Ayanath Kuttiyatveetil A Thesis Submitted to the College of Graduate Studies and Research University of Saskatchewan In Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy Department of Chemistry, University of Saskatchewan, Saskatoon, SK, Canada © Jijin Raj Ayanath Kuttiyatveetil, May 2016. All rights reserved. Permission to Use In presenting this thesis in partial fulfillment 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 work was completed. 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 this 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 Pyranose-Furanose mutases are enzymes that catalyze the isomerization of six-membered pyranose and five-membered furanose forms of a nucleotide-based sugar. In this research, the substrate binding site of three different mutases were investigated; UDP-galactopyranose mutase (UGM), GDP-altro-heptopyranose mutase (GaHM) and UDP-arabinopyranose mutase (UAM). Both UGM and UAM use a UDP-based sugar as the substrate but require different cofactors, flavin adenine dinucleotide (FAD) and Mn2+ respectively, to function. UGM and GaHM use the same cofactor (FAD), but the latter prefers to work with a GDP-based sugar. In this thesis, studies have been conducted on these three mutases using a variety of tools, such as X-ray crystallography, protein modeling, site-directed mutagenesis and kinetic assays, to understand how these enzymes bind their respective substrates. Among these three mutases, UGM is the best-studied enzyme and is a validated drug target in Mycobacteria. Despite this, the structural role of some active site residues in substrate binding is not clearly understood. Deinococcus radiodurans UGM (DrUGM) mutants of active site residues Trp184, Arg364, His88, and Asn372 were prepared and evaluated using kinetic and docking studies. The results suggested that these residues are vital to the positioning of UDP- galactopyranose under FAD in a productive conformation, for maximum enzyme efficiency. Inhibition studies, using the inhibitor MS-208, were performed on Mycobacterium tuberculosis UGM (MtUGM). Kinetic assays indicated that MS-208 is a mixed-type inhibitor of MtUGM. In this study, the crystal structures of Campylobacter jejuni GaHM (CjGaHM) with a substrate mimic GDP-mannose were solved, allowing for a comparison of GaHM and UGM substrate binding sites. The results highlighted the alterations undergone by CjGaHM to accommodate a GDP-based substrate in the active site. ii A preliminary model of UAM was built based on the protein sequence of Oryza sativa UAM1 (OsUAM1) using the protein structure modeling servers I-TASSER and GalaxyWEB. The models suggested that, unlike the catalytic role played by the FAD cofactor in UGM and GaHM, the role of the Mn2+ cofactor in UAM could be to aid the stabilization of the negative charge of the substrate diphosphate. Furthermore, experiments with mutants of OsUAM1 have helped identify residues that may bind the metal cofactor. iii Acknowledgments Firstly, I would like to thank my supervisor, Dr. David Sanders, for giving me the opportunity to carry out research in his laboratory and providing an excellent and flexible environment to learn and work. I thank him for the support and guidance, during my Ph.D. program at the University of Saskatchewan. I feel greatly indebted to him for his patience and ever- optimistic attitude, which helped me immensely through tough times. Secondly, I would like to thank Dr. David Palmer, for his suggestions and timely help in my project work. I have gained greatly from his knowledge and guidance. I am thankful to all other members of my advisory committee, Dr. Dale Ward and Dr. Miroslaw Cygler, whose inputs have enabled my project progress. I am very grateful to the Department of Chemistry for the support during my stay and study. I have had the good fortune of working with and learning from a number of people during my program. I would like to thank Dr. Karin van Straaten, for her suggestions in conducting research and help in getting familiar with the data collection at CLS and data processing. I want to extend my gratitude to other past members of the Sanders lab, Dr. Sean Dalrymple, Dr. Karunan Partha Sarathy and Dr. Josiah Obiero for teaching me various laboratory techniques and sharing scientific ideas. I want to record my appreciation to all collaborators, Dr. Julien Cotelesage, Department of Geology, University of Saskatchewan, Dr. Todd Lowary, University of Alberta and Dr. Mario Pinto, Simon Frazer University for their contributions towards my research. I would like to thank all current members of the Sanders lab for creating a great working environment. A special word of thanks to Naheda Sahtout, for her help with my thesis. I also want to take this opportunity to thank all past and present members of the Palmer lab, whom I have had the pleasure of knowing at a personal and professional level. Last but not the least, I thank all my iv friends, who have supported me in different circumstances during my stay in Saskatoon. I also thank my family members for their constant encouragement without which I would not be where I am today. v Table of Contents Permission to Use ............................................................................................................................ i Abstract ........................................................................................................................................... ii Acknowledgments.......................................................................................................................... iv Table of Contents ........................................................................................................................... vi List of Tables ................................................................................................................................. xi List of Figures ............................................................................................................................... xii List of Abbreviations .................................................................................................................... xv Chapter 1: Introduction ................................................................................................................... 1 1.1 Importance of furanoses in cell walls of organisms .............................................................. 1 1.2 D-Galactofuranose ................................................................................................................ 2 1.4 L-Arabinofuranose ................................................................................................................ 6 1.4.1 Biosynthesis of L-Arabinofuranose in plants ................................................................. 6 1.5 6d-D-altro-heptofurnaose...................................................................................................... 7 1.6 Precursors of furanoses ......................................................................................................... 8 1.7 Pyranose-furanose mutases ................................................................................................... 9 1.7.1 UDP-galactopyranose mutase......................................................................................... 9 1.7.2 Mechanism of UDP-galactopyranose mutase ............................................................... 10 1.7.3 Structure of prokaryotic UDP-galactopyranose mutases.............................................. 13 1.7.4 Structure of eukaryotic UDP-galactopyranose mutases ............................................... 16 1.7.5 Comparison between prokaryotic and eukaryotic UDP-galactopyranose mutases - mobile loop flexibility ........................................................................................................... 18 1.7.6 Mutation and modeling studies on UDP-galactopyranose mutases ............................. 20 1.7.7 Inhibitors of UDP-galactopyranose mutase .................................................................. 21 1.8 Active sites of prokaryotic and eukaryotic UDP-galactopyranose mutase ......................... 22 1.9 Other pyranose-furanose mutases ....................................................................................... 35 1.9.1 UDP-arabinopyranose mutase ...................................................................................... 37 1.9.2 GDP-6d-altro-heptopyrnaose Mutase .......................................................................... 41 vi 1.10 Objectives of the Research