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Investigating the Role of ADP-Forming Acetyl-Coa Synthetase from The Clemson University TigerPrints All Dissertations Dissertations 5-2016 Investigating the Role of ADP-forming Acetyl-CoA Synthetase from the Protozoan Parasite Entamoeba histolytica Cheryl Page Jones Clemson University, [email protected] Follow this and additional works at: https://tigerprints.clemson.edu/all_dissertations Recommended Citation Jones, Cheryl Page, "Investigating the Role of ADP-forming Acetyl-CoA Synthetase from the Protozoan Parasite Entamoeba histolytica" (2016). All Dissertations. 1668. https://tigerprints.clemson.edu/all_dissertations/1668 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]. INVESTIGATING THE ROLE OF ADP-FORMING ACETYL-COA SYNTHETASE FROM THE PROTOZOAN PARASITE 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 Cheryl Page Jones May 2016 Accepted by Dr. Cheryl Ingram-Smith, Committee Chair Dr. William Marcotte, Jr. Dr. Julia Frugoli Dr. Kimberly Paul ABSTRACT ADP-forming acetyl-CoA synthetase (ACD; EC 6.2.1.13) catalyzes the reversible conversion of acetyl-CoA to acetate coupled to the production of ATP. This enzyme is present only in certain acetate-producing archaea and a limited number of bacteria and eukaryotes. ACD belongs to the same NDP-forming acyl-CoA synthetase enzyme superfamily as succinyl-CoA synthetase (SCS; EC 6.2.1.4) from the citric acid cycle, and a similar three-step mechanism involving a phosphoenzyme intermediate was originally proposed for this enzyme. ACD has been postulated to be a major acetate-producing enzyme in the protozoan parasite Entamoeba histolytica and may contribute to ATP production. Biochemical and kinetic characterization of recombinant E. histolytica ACD (EhACD) revealed that this enzyme may function in the direction of acetate production for generation of ATP and CoA during growth in the high glucose environment of the small intestine, and in acetate assimilation to acetyl-CoA in the high acetate environment of the lower intestine during colonization. EhACD utilizes multiple substrates including propionate and propionyl-CoA supporting an additional proposed role in amino acid degradation. EhACD activity is regulated by both ATP and PPi, important energy molecules in E. histolytica. The ACD mechanism has been controversial, as a required second phosphorylation step was proposed for the Pyrococcus furiosus enzyme. Investigation of the catalytic role of the two proposed phosphorylation sites in EhACD revealed that His252, the site of phosphorylation in the original three-step mechanism, is essential for ii activity and His533, the proposed second phosphorylation site, is important but not essential. Likewise, Glu213, proposed to play a role in phosphorylation/ dephosphorylation of His252, is also required but Asp674 thought to stabilize the phosphohistidine is not. These results suggest that EhACD follows a three-step mechanism with a single phosphoenzyme intermediate. Additional conserved active site residues were examined for their role in catalysis. Asp314 was shown to be essential for activity, possibly in both a catalytic role and a structural role. Alteration at this position resulted in complete loss of activity, and computational modeling based on the Candidatus Korarchaeum cryptofilum ACD-I structure suggests that this residue may be critical for dimerization. Future directions for understanding the complex mechanism of ACD and its physiological role are presented. iii DEDICATION I would like to dedicate this work to my late mother, Priscilla Ann Mills Howell. You always have and always will be my greatest inspiration in life. My dreams are your dreams. My accomplishments are because of you. To my uncle, Charles Mills – thank you for everything. My education would not have been possible without your kind soul and generous heart. To my father, Lewis Howell – thank you for gifting me with the stubbornness to succeed. And to the rest of my family who are too numerous to name. I love you all. iv ACKNOWLEDGEMENTS First, I’d like to acknowledge my advisor Dr. Cheryl Ingram-Smith. Thanks to fortuitous timing, I had the opportunity and pleasure of being your first graduate student. I’ve always felt that our pairing was meant to be, and I’ve learned a great deal about science, hard work, and life through this experience. You’ve played a tremendous role in my growth as a scientist and a professional. I can’t wait to make you proud in the future. I’d also like to acknowledge my committee members for the immeasurable support throughout this process. Thanks to Dr. Julia Frugoli for encouraging me to pursue the NSF fellowship. Aside from the financial support, the confidence the fellowship has given me was instrumental in my success. Thanks to Dr. Kimberly Paul for always having an open door and letting me hijack your classroom. Thanks to Dr. William Marcotte for letting me carve my own unconventional path at times. Thank you all for the extra support in pursuing my education certificate. A special thanks goes to Dr. Kerry Smith and the Smith lab for constant support and collaboration. Without your advice (and many pieces of equipment), this project would not have been the same. Furthermore, all of the members of the Eukaryotic Pathogens Innovation Center have helped me in one way or another and made this journey truly enjoyable and entertaining. I thank my fellow Ingram-Smith Lab graduate student, Thanh Dang, who has been a sounding board for all of my theories and ideas; Katie Glenn and Tonya Taylor for being excellent teachers and friends; and my fellow Entamoeba comrades, particularly Hollie Hendrick and Brenda Welter. v Two wonderful undergraduate students, Kirin Khan and Noah Smith, have directly contributed to this work. Thank you for the opportunity to be a mentor and the hard work you’ve invested in this project. And finally, the National Science Foundation and the College of Agriculture, Forestry, and Life Sciences, that have provided me with three excellent fellowships during my tenure as a graduate student. vi TABLE OF CONTENTS PAGE TITLE PAGE .................................................................................................................... i ABSTRACT .................................................................................................................... ii DEDICATION ................................................................................................................ iv ACKNOWLEDGEMENTS ............................................................................................... v LIST OF TABLES .......................................................................................................... ix LIST OF FIGURES ......................................................................................................... x CHAPTER 1. LITERATURE REVIEW ..................................................................................... 1 I. Introduction ................................................................................ 1 II. Acetate Metabolism .................................................................... 2 III. NDP-forming Acyl-CoA Synthetases ........................................ 14 IV. Entamoeba histolytica .............................................................. 24 V. Conclusions .............................................................................. 35 VI. References ............................................................................... 36 2. BIOCHEMICAL AND KINETIC CHARACTERIZATION OF THE RECOMBINANT ADP-FORMING ACETYL-COA SYNTHETASE FROM THE AMITOCHONDRIATE PROTOZOAN ENTAMOEBA HISTOLYTICA ................................................................................................ 53 I. Abstract .................................................................................... 53 II. Introduction .............................................................................. 55 III. Materials and Methods ............................................................. 59 IV. Results ..................................................................................... 64 V. Discussion ................................................................................ 74 VI. Acknowledgements .................................................................. 81 VII. References ............................................................................... 82 3. INVESTIGATING THE MECHANISM OF ADP-FORMING ACETYL-COA SYNTHETASE FROM THE PROTOZOAN PARASITE ENTAMOEBA HISTOLYTICA ....................................................... 86 I. Abstract .................................................................................... 86 vii Table of Contents (Continued) II. Introduction .............................................................................. 88 III. Materials and Methods ............................................................. 93 IV. Results ..................................................................................... 99 V. Discussion .............................................................................. 108 VI. Acknowledgements ................................................................ 114 VII. References ............................................................................
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