DEFINING THE CATALYTIC AND KINETIC MECHANISM AND NATURAL FUNCTION OF THE HIGHLY CONSERVED ACYL-AMP HYDROLASE, HINT1 A DISSERTATION SUBMITTED TO THE FACULTY OF THE GRADUATE SCHOOL OF THE UNIVERSITY OF MINNESOTA BY SANAA BARDAWEEL IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTORE OF PHILOSOPHY DR. CARSTON R. WAGNER, ADVISER DECEMBER 2010 © Sanaa Bardaweel, 2010 ACKNOWLEDGEMENTS I would like to gratefully and sincerely thank my thesis adviser, Dr. Carston R. Wagner, for his guidance, understanding and patience. His mentorship was paramount in providing a well rounded experience consistent with my long-term career goals. He encouraged me to not only grow as an experimentalist but also as an instructor and an independent researcher. In every sense, none of this work would have been possible without him. I gratefully acknowledge Dr. Michael Sadowsky for his advice, supervision, and crucial contributions, which made him a backbone of this research and so to this thesis. I am also deeply thankful to Dr. Patrick Hanna, for the valuable advice and discussions that inspired me through out the work. Many thanks must also go to my thesis committee members, Dr. Chengguo Xing and Dr. Howard Towle, for their valuable input and suggestions that improved my dissertation. I would like to make a special reference to Dr. Yusuf Abul-Hajj for his unflinching encouragement and support in various ways. His support and guidance during the very first steps in my PhD have had a remarkable influence on my entire progress in the program. I am grateful to former members of Dr. Wagner’s laboratory, Dr. Brahma Ghosh, Dr. Brandie Brummer, Dr. Yan Jia, Dr. Qing Li, Dr. Brian White and Dr. Xin Zhou for their help and great friendship. I also thank current members of the lab, Dr. Adrian Fegan, Dr. Matthew Cuellar, Dr. Sidath Kumarapperuma, Amit Ganger and Ryan Holton for the numerous and productive discussions that helped me improve my knowledge during my graduate study. i I owe my loving thanks to my mother who raised me with a love of science and supported me in all my life. Her endless faith inspired me to become the person who I am today. A special thank-you goes to my wonderful sons, Hashem, Yamen and Othman who made my graduate life a lot less stressful. I hope that I may serve as an example for them as my mother has for me. My final, and most heartfelt, acknowledgment must go to my husband Rizq. His support, encouragement and unwavering love have turned my journey through graduate school into a pleasure. For never once complaining through all difficult times and challenges, for taking all responsibility for the care of our family, for sharing all the good and bad times with endless support, for all that, this dissertation is dedicated to him. ii ABSTRACT Histidine triad nucleotide binding proteins (Hints) are members of the histidine triad (HIT) protein superfamily of nucleotidyl transferases and hydrolyases. It has been recently demonstrated that Hints are efficient phosphoramidases and therefore activators of potent antiviral and anticancer pronucleotides. In spite of their high evolutionary conservation among all kingdoms of life, and the several regulatory functions in which Hints have been implicated, a clear connection between their observed function and their catalytic efficiency has not been elucidated. To gain a comprehensive understanding of the essential role of these ubiquitous enzymes, our laboratory has devoted a considerable effort toward the delineation of the principles governing Hints catalysis and cellular function. Such understanding will provide an unprecedented ability to assess the role of these highly conserved, but functionally unknown enzymes. Since Hints are found in both prokaryotes and eukaryotes, we have attempted to understand their function, mechanism, and structural determinants in prokaryotes, under the assumption that their role may be at least partially conserved among members of the tree of life. Recently, we have demonstrated by E. coli gene disruption studies that the bacterial Hint enzyme is necessary for growth under high salt conditions, and when alanine is a carbon and nitrogen source. Through a combination of phenotypic screening and complementation experiments with wild-type and ecHinT knock-out E. coli strains, we have shown that catalytically-active ecHinT is required for growth on D-alanine. In addition, using Hint-inhibitors and active-site mutants, we have demonstrated that expression of catalytically-active ecHinT is essential for the activity of the enzyme D- amino acid dehydrogenase (DadA) (equivalent to D-amino acid oxidase in eukaryotes), a iii necessary component of the D-amino acids metabolic pathway. These results are considered as the first report in literature that shows a successful connection between a discovered Hint-related phenotype and the catalytic activity of Hint. Previously, we have demonstrated that lysyl-AMP generated by LysRS is a substrate for both human and E. coli Hints. In addition, we have shown that the ability of Hint to hydrolyze lysyl-AMP depends on its enzymatic activity. Here, we demonstrate that the molecular determinants governing this regulation appear to reside in the C- terminus region of Hint. Interestingly, the ecHinT-DadA interaction appears also to be governed by both ecHinT-activity and the C-terminus loop. We have also expanded our scope to look at possible toxicity of D-alanine in E. coli strains lacking dadA or hinT . Our results demonstrate that E. coli mutants lacking dadA or hinT are highly susceptible to D-alanine toxicity and that the catalytic activity of Hint is an essential requirement to protect E. coli from the observed toxicity of D-alanine. Based on careful analysis of the combined results from the ecHinT-LysRS and ecHinT- DadA potential interactions, and comprehensive understanding of the D-alanine metabolic pathway in bacteria, we proposed a possible regulatory mechanism of Hint, LysRS and DadA on global protein translational processes to prevent D-amino acids toxicity in E. coli . iv Table of Contents Acknowledgements ............................................................................................................ i Abstract ............................................................................................................................. iii Table of Contents ...............................................................................................................v List of Tables .................................................................................................................... xi List of Figures ................................................................................................................. xiii List of Schemes ............................................................................................................... xvi List of Abbreviations .................................................................................................... xvii Chapter One: Introduction: Histidine Triad Nucleotide Binding Proteins .................1 I. Discovery and Classification of HIT Superfamily...................................................2 II. Phosphoramidase Activity of Hints .........................................................................6 III. Structural Properties of Hints ................................................................................12 IV. Structural Studies of Hints ....................................................................................13 V. Role of Hint Homodimerization ...........................................................................14 VI. Possible Biological Functions of Hints ..................................................................18 VII. Prokaryotic Hint ....................................................................................................20 VIII. The Interaction of Hints with Aminoacyl tRNA Synthetases ...............................23 IX. Current Research ...................................................................................................27 Chapter Two: Probing the Impact of the ecHinT C-Terminal Domain on Structure and Catalysis.....................................................................................................................28 v I. Introduction ............................................................................................................29 II. Experimental Procedures .......................................................................................32 A. Crystallization and X-ray Data Collection .............................................................32 B. Structure Determination and Refinement ..............................................................32 C. Site-directed Mutagenesis ......................................................................................35 D. Expression and Purification of Recombinant Proteins ..........................................35 E. Circular Dichroism Spectroscopy ..........................................................................37 F. Size-exclusion Chromatography ............................................................................37 G. Steady-state Kinetics ..............................................................................................38 H. Pre-steady-state Kinetics ........................................................................................38 I. pH-dependence of Steady-state Kinetics ...............................................................39