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On the Preorganization of the Active Site of Choline Oxidase for Hydride Transfer and Tunneling Mechanism

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Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry 6-23-2009 On the Preorganization of the Active Site of Choline Oxidase for Hydride Transfer and Tunneling Mechanism Osbourne Quaye Georgia State University Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Part of the Chemistry Commons Recommended Citation Quaye, Osbourne, "On the Preorganization of the Active Site of Choline Oxidase for Hydride Transfer and Tunneling Mechanism." Dissertation, Georgia State University, 2009. https://scholarworks.gsu.edu/chemistry_diss/46 This Dissertation is brought to you for free and open access by the Department of Chemistry at ScholarWorks @ Georgia State University. It has been accepted for inclusion in Chemistry Dissertations by an authorized administrator of ScholarWorks @ Georgia State University. For more information, please contact [email protected]. ON THE PREORGANIZATION OF THE ACTIVE SITE OF CHOLINE OXIDASE FOR HYDRIDE TRANSFER AND TUNNELING MECHANISM by OSBOURNE QUAYE Under the Direction of Giovanni Gadda ABSTRACT Choline oxidase catalyzes the two-step oxidation of choline to glycine betaine, one of limited osmoprotectants, with the formation of betaine aldehyde as an enzyme bound intermediate. Glycine betaine accumulates in the cytoplasm of plants and bacteria as a defensive mechanism to withstand hyperosmolarity and elevated temperatures. This makes the genetic engineering of relevant plants which lack the property of salt accumulation of economic interest, and the biosynthetic pathway of the osmolyte a potential drug target in microbial infections. The reaction of alcohol oxidation occurs via a hydride ion tunneling transfer from the substrate donor to a flavin acceptor within a highly preorganized active site environment in which choline and FAD are in a rigidly close proximity. In this dissertation, factors contributing to the enzyme-substrate preorganization which is required for the hydride ion tunneling reaction mechanism in choline oxidase have been investigated. Crystallographic studies of wild-type choline oxidase revealed a covalent linkage between C8M atom of the FAD isoalloxazine ring and the N(3) atom of the side chain of a histidine at position 99, and a solvent excluded cavity in the substrate binding domain containing glutamic acid at position 312 as the only negatively charged amino acid residue in the active site of the enzyme. The role of the histidine residue and the contribution of the 8α-N(3)-histidyl covalent linkage of the flavin cofactor to the reaction of alcohol oxidation was investigated in a variant form of choline oxidase in which the histidine residue was replaced with an asparagine. The role of the glutamate residue and the importance of the spatial location of the negative charge at position 312 was investigated in variant forms of choline oxidase in which the negatively charged residue was replaced with glutamine and aspartate. Mechanistic data obtained for the variant enzymes and their comparison to previous data obtained for wild-type choline oxidase are consistent with the residues at positions 99 and 312 being important for relative positioning of the hydride ion donor and acceptor. The residues are important for the enzyme-substrate preorganization that is required for the hydride tunneling reaction in choline oxidase. INDEX WORDS: Flavoproteins, Flavinylation, Quantum Mechanical Tunneling, Hydride Ion Transfer, Preorganization, Choline Oxidase, Alcohol Oxidizing Enzymes ON THE PREORGANIZATION OF THE ACTIVE SITE OF CHOLINE OXIDASE FOR HYDRIDE TRANSFER AND TUNNELING MECHANISM by OSBOURNE QUAYE A Dissertation Submitted in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in the College of Arts and Sciences Georgia State University 2009 Copyright by Osbourne Quaye and Giovanni Gadda 2009 ON THE PREORGANIZATION OF THE ACTIVE SITE OF CHOLINE OXIDASE FOR HYDRIDE TRANSFER AND TUNNELING MECHANISM by OSBOURNE QUAYE Committee Chair: Dr. Giovanni Gadda Committee: Dr. Dabney W. Dixon Dr. Yujun G. Zheng Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University August 2009 iv To God Be The Glory & Dedicated To All Who Draw Inspiration From Me To Achieve Their Personal Academic Goals v ACKNOWLEDGEMENTS In as much as completion of my Ph.D. is obviously a great feat, it is not an accomplishment that I can solely take credit for. Just like a newborn child growing up, my study started with baby steps and needed the watch and support of many in order to develop the necessary skill and discipline to achieve the goal of becoming a seasoned scientist. It is with immeasurable appreciation that I acknowledge my program advisor, Dr. Giovanni Gadda. Even though I have sometimes honestly felt the “knocks” were unjustifiably too hard, your unwavering support and “push” is what has really taken me to the “Olympics”. If I made it all the way through, it is because you made it all happen. Over the years as your student, my attitude to science and research, as well as working life in general, has tremendously changed for the better; not to mention the confidence you have inadvertently imbibed in me as a result of your knowledgeable prowess and pedigree in your field of research. The disciplined training I have acquired under your tutelage and direction will forever remain a tall-standing fortress in my life in the future. Thank you very much for the crafty touch you gave my career. My second appreciation goes to my advisory committee members, Drs. Dabney W. Dixon and Yujun G. Zheng, for your prompting questions and suggestions, and the fine tunings of some of the rough edges of my study. Dr. Dixon, I would like you to exceptionally know that your motherly and career goals talk/advice are not only going to be used right after my program but for a lifetime. My appreciation also goes to all the professors who in one way or the other taught or offered me advice in my Ph.D. studies, both in Georgia and Ghana. To my academic god-parents, Professor and Professor Mrs. Ernest and Ellen Aryeetey, Professor Marian E. Addy, and Professor Alex Nyarko, I say thank you and I am particularly highly appreciative of you for believing in me. I also want to take this opportunity to thank Professor Nigel Scrutton, Dr. Sam vi Hay, Dr. Chris Pudney and all the members of Nigel Scrutton’s laboratory at the University of Manchester, UK for hosting me when I came to do part of my study in Manchester. There is always something to learn from people however brief the interaction with them may be. It is and would forever be a wonderful experience getting to meet and working with all the past and present members of Dr. Giovanni Gadda’s laboratory that I met. Fan, Mahmoud, Kevin, Trang, Tran Bao, Kunchala, Merid, Steffan, Slavica, Andrea, Anthony, Hongling, Sharonda, Lydia, Philip, Nicole, Stephen and Danila you all provided the serene laboratory environment in your own individual ways for this successful Ph.D. story to be told. Tore (Kunchala), for all the enjoyment and fun I had in your black Toyota Camry, I have considered owning a Toyota Camry in the future; you are such a sweet-hearted angel. I would also like to mention the cheerfully receiving faces of the staff at the Chemistry Department of Georgia State University. Edna, Sina, Stephanie, Diane, Marina, Lita, Robert, Will, Dan, Chris Long (My Hero), Don Hardon etc. your smiles and willingness to always provide the needed help were so soothing they will never be forgotten. To Julianne, Cathy, Rong, Jia and all of my student friends, thank you for being special to me. I am indebted to my wife Naa and daughter Gift for being the backbone that kept me going whenever the going got tough. I denied you your rightfully deserving time and attention during my Ph.D. study. Naa, I left you in the mornings almost everyday (at least every weekday) to take care of Gift and the home, and came back late in the evening too tired to even help you with anything. You took care of me and even picked up after me as if I were a baby too, just so I could find the time to do my school work. For all the sacrifice you had to make, no amount of “Thank you” I say and in whatever language I say it will suffice. I equally share the honors with vii you and more than ready to make it up to you in every way possible. I pledge my love to you and I pray God richly bless you. I would like to say “Thank you” to my parents Poppyyy and Mommyyy, my grandma Yoomo, Mama Belinda, all my siblings, and my entire extended family for your unending love to me. I feel so blessed for all the emotional support from the rank and file of the whole family. Without all of you, there would have been no me. To my friends David and Rosemond, Ben and Amma, Serwaa and Kufour, Heather, Selina, Josephine, Andy Andoh, and Abena Ajobi all of Atlanta, GA, Adriana of West Orange, NJ, Norine, Ernest, Joseline and Jonathan, I appreciate you being there when it mattered most during my Ph.D. program. My final thanks go to all those who could not be mentioned; your contributions and support are very much appreciated and are by no means less of importance. Thank you once again to all and sundry and God richly bless each and every one of you. viii TABLE OF CONTENTS ACKNOWLEDGEMENTS .............................................................................................................v LIST OF TABLES .......................................................................................................................
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