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BENZOYL-COA REDUCTASE

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ABSTRACTTitle of Dissertation: BENZOYL-COA REDUCTASE: A BIOLOGICAL BIRCH REDUCTIONSteven Thomas Poole, Doctor of Philosophy, 2003Dissertation directed by: Dr. David R. JollieDepartment of ChemistryBenzoyl-CoA reductase, isolated from the anaerobic bacterium Thaueraaromatica, catalyzes the ATP-dependent, two-electron reduction of the aromaticring of benzoyl-CoA. A Birch-like mechanism, which involves two separate oneelectron and one proton additions to the aromatic ring of benzoyl-CoA, has beenpreviously proposed for benzoyl-CoA reductase. The first electron transfer of thisreaction, which produces a radical anion, is thought to be the rate-limiting step. Other mechanisms, such as hydride reduction and catalytic hydrogenation, arepossible. In an effort to determine how the enzyme reduces its substrate, severalsubstrate analogues were synthesized and studied using kinetic and/or productanalysis. Of the nitrogen-containing, heterocyclic analogues, only picolinoyl-CoAproved to be a substrate for the reductase, having a kcat similar to that of benzoyl-CoA. Nicotinoyl-CoA did not react with the enzyme and isonicotinoyl-CoA wasreduced by the electron donor in the absence of the enzyme. Mass spectrometricanalysis of the products formed by the fluorinated analogues, m-fluorobenzoyl-CoA and p-fluorobenzoyl-CoA indicated that both substrates were defluorinated bybenzoyl-CoA reductase, supporting a Birch-like mechanism with the first electronbeing added to the carbonyl functionality of the thioester. Also, benzoyl-CoAreductase only exhibited a small kinetic isotope effect (1.8), arguing againstsimultaneous hydrogen and electron transfer and hydride transfer. It was alsofound that under aerobic conditions and without ATP, benzoyl-CoA reductasecould carry out the oxidation of its native reduction product reforming the substrateof the reaction, benzoyl-CoA. Since the reduction capability of benzoyl-CoAreductase is quickly and irreversibly inactivated by oxygen, it is thought that theenzyme is degraded under aerobic conditions. However, these finding suggest thatbenzoyl-CoA reductase may only be partially degraded by oxygen exposure andthat some of its subunits may still retain some of its functionality and structure.BENZOYL-COA REDUCTASE: A BIOLOGICAL BIRCHREDUCTIONbySteven Thomas PooleDissertation submitted to the Faculty of the Graduate School of theUniversity of Maryland, College Park in partial fulfillmentof the requirements for the degree ofDoctor of Philosophy2003Advisory Committee:Professor Steven E. Rokita, ChairDr. David R. Jollie, Co-chair/AdvisorProfessor Dorothy BeckettProfessor J. Norman HansenProfessor Douglas A. JulinProfessor Richard C. StewartiiDEDICATIONThis dissertation is dedicated to my family for their constant support and tomy advisor, Dr. David Jollie, for his patience and guidance.iiiACKNOWLEDGEMENTSI would like to thank my advisor, Dr. David Jollie, for his helpfulinstruction, his patience, his valuable input when I ran into difficulties, and fordealing with a lot of the computer-related problems in the laboratory. Everything Ihave learned about the OS2 operating system are thanks to him. Special thanks aredue to Terry Chase, Ben Woodard, and to the staff of the BioProcess Scale-UpFacility who donated their time, assistance, and the use of their facilities for myfermentations. I would have never obtained the protein quantities needed for thisresearch without their help. I am grateful to Dr. Yui-fai Lam for his assistancewith obtaining the proton magnetic resonance spectra of the benzoyl-CoAanalogues and to Dr. Judd O. Nelson for his help with the LC/MS analyses. Iwould like to thank Professor Jason D. Kahn and Professor Steven E. Rokita fortaking the time to answer my numerous questions and for keeping me pointed inthe right direction. I would also like to thank the members of my advisorycommittee for their valuable time.I would like to specially thank Ms. Emily Luckman for lending a muchappreciated helping hand with the everyday chores in the laboratory and I wouldivlike to thank Mr. Naciem Yousif for helping to make the laboratory a little lessquiet. I would also like to thank my loving wife, Nicci, for her undending supportand for listening to my numerous practice talks..vTABLE OF CONTENTSList of Tables ................................................... viiList of Figures .................................................. viiiList of Abbreviations ............................................. xivChapter I. Background and Theoretical Rationale ........................ 1Background ............................................... 1Impact of Aromatic Compounds on the Environment .......... 1Aerobic Metabolism of Aromatic Compounds ............... 2Anaerobic Metabolism of Aromatic Compounds .............. 4The Birch Reduction ................................... 9The Components of Benzoyl-CoA Reductase ............... 11Characteristics of Fe-S Cluster-Containing Proteins .......... 12Mechanism of the Activase/2-Hydroxyglutaryl-CoA Dehydratase System of Acidaminococcus fermentans ... 13Proposed Mechanism for Benzoyl-CoA Reductase ........... 16Energetics of the “Biological Birch Reduction” .............. 18Theoretical Rationale ....................................... 20Possible Mechanisms for Aromatic Ring Reduction .......... 20Use of Substrate Analogues to Study the Mechanism of Benzoyl-CoA reductase ......................... 23Reversibility of Electron Transfer by Benzoyl-CoA Reductase .................................... 25Chapter II. Materials and Methods .................................. 27Materials and Bacterial Strain ................................. 27Growth of Bacterial Cells .................................... 27Harvesting of Bacterial Cells ................................. 28Preparation of Cell Extracts .................................. 28Purification of Benzoyl-CoA Reductase ......................... 29SDS/PAGE and Bradford Assay .............................. 30viAssay of Benzoyl-CoA Reductase Activity ....................... 31Synthesis and Purification of Coenzyme A Thioesters ............... 33Enzymatic Studies on Benzoyl-CoA Reductase Using Thioester Analogues ......................................... 36Determination of the Catalytic Properties of Benzoyl-CoAReductase .......................................... 36Separation of the Products of the Benzoyl-CoA Reductase Catalyzed Reduction of Benzoyl-CoA and Studies on the


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