Clemson University TigerPrints All Dissertations Dissertations 8-2017 Acetate Metabolism: The physiological role of ADP-forming acetyl-CoA synthetase and acetate kinase in Entamoeba histolytica Thanh Dang Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Recommended Citation Dang, Thanh, "Acetate Metabolism: The hp ysiological role of ADP-forming acetyl-CoA synthetase and acetate kinase in Entamoeba histolytica" (2017). All Dissertations. 1976. https://tigerprints.clemson.edu/all_dissertations/1976 This Dissertation is brought to you for free and open access by the Dissertations at TigerPrints. It has been accepted for inclusion in All Dissertations by an authorized administrator of TigerPrints. For more information, please contact [email protected]. Acetate Metabolism: The Physiological Role of ADP-forming Acetyl-CoA Synthetase and Acetate Kinase in Entamoeba histolytica _____________________________________________________________________ A Dissertation Presented to the Graduate School of Clemson University _____________________________________________________________________ In Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy Biochemistry and Molecular Biology _____________________________________________________________________ by Thanh Dang August 2017 _____________________________________________________________________ Accepted by: Cheryl Ingram-Smith, Committee Chair James C. Morris Kerry S. Smith Lesly Temesvari ABSTRACT Entamoeba histolytica is a protozoan parasite that causes amoebic colitis and liver abscess in approximately 90 million people each year, resulting in 50,000-100,000 fatalities. Even though Entamoeba poses a significant public health problem worldwide, research dedicated to understanding the biology of this unique protozoan has been limited. This amitochondriate parasite lacks many essential biosynthesis pathways including the tricarboxylic acid (TCA) cycle and oxidative phosphorylation. As a result, substrate level phosphorylation plays a necessary role in ATP production. Unlike the standard glycolytic pathway, E. histolytica glycolysis requires pyrophosphate (PPi) by replacing ATP–dependent phosphofructokinase and pyruvate kinase with PPi–dependent phosphofructokinase and phosphate pyruvate dikinase. E. histolytica infects and colonizes the human colon where glucose is limited and short chain fatty acids (acetate, propionate, and butyrate) are plentiful. Acetate is also a major end product that is excreted when E. histolytica is grown axenically on glucose. Acetate has been demonstrated to act as carbon and energy source for cellular growth in other organisms, acetogenesis can regenerate NAD+, recycle coenzyme A, and produce ATP when the TCA cycle or oxidative phosphorylation does not operate or when the carbon flux into the cell exceed its capacity. In E. histolytica, acetate can be generated by acetate kinase (ACK) and ADP– forming acetyl–CoA synthetase (ACD). ACK converts acetyl phosphate + orthophosphate (Pi) to acetate + PPi. Previous biochemical and kinetic characterization of II recombinant ACK showed that it strongly prefers the acetate/PPi-forming direction. We hypothesized that ACK may function to supply PPi for the PPi oriented glycolytic pathway in E. histolytica. Recombinant ACD displayed high activity in both directions of the reaction to convert acetyl-CoA + orthophosphate + ADP to acetate + ATP + CoA. ACD may function to extend the glycolytic pathway to increase ATP production by 40% per molecule of glucose, or in the alternative direction to convert acetate to acetyl-CoA to meet the cell’s metabolic needs. Using reverse genetics, an Ehacd silenced strain displayed a growth defect in normal high glucose media, while Ehack silenced cells showed enhance growth in medium without added tryptone and glucose. The presence of acetate and butyrate showed no effect on E. histolytica growth in the absence of glucose regardless of ACK or ACD activity. The presence of propionate, however, improved E. histolytica growth and impaired growth of the Ehacd silenced strain implicated ACD as the cause of this improvement. Our data suggest ACD plays a role in increasing ATP production during growth on glucose and utilization of propionate as a growth substrate. Our data do not support the previously hypothesized role for ACK but instead suggest it possesses a novel function. The basis for E. histolytica ACK’s divergence from all other ACKs in phosphoryl substrate utilization was also explored. Currently, E. histolytica ACK is the only known ACK that uses pyrophosphate (PPi) or inorganic phosphate (Pi) as the phosphoryl donor or acceptor. All other known ACKs utilize ATP or ADP. In silico structural comparison and modeling of E. histolytica ACK against other ACKs identified structural differences III that could affect substrate binding and selection. ACK variants were generated to test these predictions. Inhibition and structural activity relationship studies revealed an occlusion in the ADENOSINE motif of E. histolytica ACK reduced ATP and ADP binding affinity. However, alterations to alleviate the constriction did not confer activity with ATP or ADP. Our results suggest controlling access of the adenosine pocket influences phosphoryl substrate binding but is not the sole determinant of enzyme activity. IV DEDICATION I would like to dedicate this to my Mom, Dad, and Joanne. Without your support and encouragement, this would not have been possible. I am blessed and thankful for having such a loving family. V ACKNOWLEDGEMENT I would like to thank my advisor, Dr. Cheryl Ingram-Smith. You are kind, patient, and understanding. I am truly grateful for the opportunity to work and learn from you. The lessons I gained here will stay with me for the rest of my career. Additionally, I would like to thank my committee members. Dr. Kerry Smith: thank you for the valuable discussions and insightful inputs during our numerous joint lab meetings. Dr. Lesly Temesvari: thank you for your advice and expertise toward my Entamoeba research. Dr. James Morris: thank you for your constant wisdom and wild ideas that have kept my perspective in check. I would also like to acknowledge the students from both the Ingram-Smith and Smith labs. Without everyone’s advice and helpful technical assistance, I would have made more mistakes, repeated more experiments, and not learned as much as I have. Thank you Cheryl Jones, Jordan Wesel, and Diana Nguyen, my fellow Ingram-Smith lab mates, for being a platform to screen new ideas and experimental designs. From the Smith lab, thank you Tonya Taylor, Katie Glenn, and Satyanarayana Lagishetty for being so welcoming and of great assistance during my graduate career. Lastly, I would like to extend a special thanks to Brenda Welter and Hollie Hendrick from Dr. Temesvari’s lab. Your assistance was critical to my work with Entamoeba histolytica. I would like to also express great thanks to the faculty, staff, and graduate students from Clemson University Genetics and Biochemistry department and the Eukaryotic Pathogens Innovation Center. In particular, I would like to thank Logan VI Crowe, Sophie Altamirano, Michael Harris, Jimmy Suryadi, and Steven Cogill for their support and advice in matters of science and life. Most of all, without everyone’s technical assistance, valuable input, and words of encouragement, graduate school would not have been as enjoyable and achievable. So, thank you! VII TABLE OF CONTENTS Page TITLE PAGE....................................................................................................................... I ABSTRACT........................................................................................................................II DEDICATION....................................................................................................................V ACKNOWLEDGEMENT ................................................................................................ VI LIST OF TABLES..............................................................................................................X LIST OF FIGURES .......................................................................................................... XI CHAPTER I. Literature Review.........................................................................................1 Introduction......................................................................................1 Acetate Metabolism .........................................................................3 Acetate metabolism in bacteria............................................4 Acetyl-phosphate in signal transduction..................9 Acetate metabolism in eukaryotes .....................................11 Entamoeba histolytica....................................................................16 Metabolism ........................................................................20 Acetate Kinase ...............................................................................28 ATP-acetate kinase ............................................................28 PPi-acetate kinase ..............................................................37 References.......................................................................................41 II. Investigation of pyrophosphate versus ATP substrate selection in the Entamoeba histolytica acetate kinase ...............................58 Abstract..........................................................................................58