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Dna Polymerase Beta, Carboxypeptidase, and Acetyl Coenzyme-A Decarbonylase/Synthase

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Dna Polymerase Beta, Carboxypeptidase, and Acetyl Coenzyme-A Decarbonylase/Synthase CHARACTERIZATION AND STRUCTURAL DETERMINATION OF METALLOENZYMES: DNA POLYMERASE BETA, CARBOXYPEPTIDASE, AND ACETYL COENZYME-A DECARBONYLASE/SYNTHASE DISSERTATION Presented in Partial Fulfillment of the Requirement for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University By Joseph W. Arndt, B.S. ***** The Ohio State University 2003 Dissertation Committee: Approved by Professor Michael K. Chan, Advisor Professor Joseph Krzycki ___________________________ Professor Ming-Daw Tsai Advisor Department of Chemistry ABSTRACT My research focused on the structure determination of proteins from three metalloenzyme systems by X-ray crystallography. The first target was rat DNA polymerase β, which catalyzes the template-directed nucleotidyl transfer reaction required for DNA replication. We have determined the crystal structures of two intermediate complexes in the reaction pathway of this enzyme, (i) a pre-chemistry ternary complex containing protein, DNA, and a chromium dNTP analog and (ii) a post- chemistry complex after nucleotide incorporation. These intermediate structures have allowed us to dissect the role of the two essential magnesium ions in initiating the enzyme’s conformational change. Based on these structures, a revised mechanism for replication and fidelity is proposed. The second part of this research involved structural studies on a carboxypeptidase (PfuCP) from the hyperthermophilic archaeon, Pyrococcus furiosus. Like other carboxypeptidases, it catalyzes the removal of amino acids from the C-terminus of protein and peptide chains. In this project we have solved three different structures of this enzyme, an apo form and two metal-bound forms. The overall fold of this enzyme is distinct from all other known structures of carboxypeptidase. It differs significantly in sequence, however, with one important feature being a consensus HEXXH metal-binding ii motif at its active site. While HEXXH motifs are common in aminopeptidases and endopeptidases, this is the first observation of this motif in a carboxypeptidase. These structures represent the first prototype available for this growing family of carboxypeptidase. Comparison with other metallopeptidases allowed us to propose a catalytic mechanism of its C-terminal peptide hydrolysis reaction. The last target of this research was the Methanosarcina barkeri acetyl coenzyme- A decarbonylase/synthase (ACDS) complex and its five individual components. The ACDS complex is involved in the metabolism acetyl coenzyme-A by methanogens. In this project we have isolated ACDS and its subunits, α2ε2, β, γ, and δ, for crystallization and subsequent structure elucidation. Several crystal forms have been identified for the α2ε2 and β components. In addition, we have performed metal chelation and reconstitution experiments on the β component that indicate the enzyme’s acetyl transferase activity is dependent on nickel, and not copper. iii Dedicated to my wife and parents iv ACKNOWLEDGMENTS I would like to express my utmost gratitude to my advisor, Dr. Michael Chan, for his generosity, encouragement, and support. The freedom for which he permitted me to perform my research allowed me to become a better scientist – thank you. I would also like to acknowledge my committee members, Drs. Tsai and Krzycki, for their wisdom, precious time, and assistance. Their passion for science is contagious. It has been an honor and my sincere privilege to work with them. I am indebted to past post docs, Drs. Weimin Gong and Xuejun Zhong, which I have had the pleasure of working with on the polymerase β project. I would like to extend my appreciation to my lab mates, Dr. Bing Hao, Clara Isaza, Rinku Jain, Patrick Kang, and Michelle Nauerth for their continual willingness to lend a hand, their technical insight, and numerous conversations that we shared together over the years. A special thanks goes out to Bing, Clara, and Rinku, not only for the scientific camaraderie that we shared, but also for their friendship and good times we spent together – my years at Ohio State were made all the more enjoyable by their company. v Particular recognition goes to Dr. Tsai and my colleagues of the Chemistry and Biology Interface Training Program funded by the NIH from which I was honored with a fellowship. And to the Graduate Studies Committee and Miles Foundation for granting me a fellowship for the last six months of my studies, which made the writing of this dissertation possible. I would like to acknowledge my cats, Pele-boy and Cashew, for kindly donating their whiskers for the cause of science - my crystal seeding experiments would have been unfruitful them. Finally, I am most thankful to my wife, parents, and mother-in-law for their unwavering love and support. This degree would not have been possible without their prayers and encouragement. vi VITA October 24, 1968……………………… Born, Chicago, IL 1987 - 1991…...………………………. B.S., Chemistry, Illinois State University, Normal, IL 1991 – 1996...………………………… Research Technician, Abbott Laboratories, North Chicago, IL 1997 - 2000…………………………… Graduate Teaching and Research Associate, The Ohio State University 2000 – 2002…………………………… NIH Predoctoral Fellowship, Chemistry and Biology Interface Program 2003 – present ………………………… Miles Fellowship, The Ohio State University vii RELATED PUBLICATIONS 1. Bing Hao, Clara Isaza, Joseph W. Arndt, Michael Soltis and Michael K. Chan (2002). Structure-based mechanism of O2 sensing and ligand discrimination by the FixL heme domain of Bradyrhizobium japonicum, Biochemistry, 41, 12952-8. 2. Joseph W. Arndt, Bing Hao, Vijay Ramakrishnan, Timothy Cheng, Sunney I, Chan and Michael K. Chan (2002). Crystal structure of a novel carboxypeptidase from the hyperthermophilic Archaeon Pyrococcus furiosus, Structure, 10, 215-24. 3. Joseph W. Arndt, Weimin Gong, Xuejun Zhong, Alexander K. Showalter, Jia Liu, Christopher Dunlap, Ming-Daw Tsai, and Michael K. Chan (2001). Insight into the catalytic mechanism of DNA polymerase β: structure of intermediate complexes, Biochemistry, 40, 5368-75. FIELDS OF STUDY Major Field: Chemistry Studies in X-ray Crystallography of Biological Macromolecules viii TABLE OF CONTENTS ABSTRACT........................................................................................................................ ii DEDICATION................................................................................................................... iv ACKNOWLEDGMENTS .................................................................................................. v VITA................................................................................................................................. vii LIST OF FIGURES .......................................................................................................... xv LIST OF TABLES........................................................................................................... xix LIST OF PDB ID.............................................................................................................. xx LIST OF ABBREVIATIONS.......................................................................................... xxi CHAPTER 1 .......................................................................................................................1 1.1 Functional variability of metals .............................................................................. 1 1.2 Metal cofactor variability........................................................................................ 2 ix 1.2.1 Magnesium-dependent enzymes..................................................................... 3 1.2.2 Cobalt- dependent enzymes ............................................................................ 5 1.2.3 Nickel-dependent enzymes ............................................................................. 6 1.2.4 Metalloclusters in biology............................................................................... 6 1.3 Metalloproteins are just “MAD” about synchrotron radiation ............................... 8 1.4 The highlight of this research ................................................................................. 8 1.4.1 Intermediate structures in catalytic pathway of DNA polymerase β.............. 9 1.4.2 Pyrococcus furiosus carboxypeptidase: crystal structure of a novel class of carboxypeptidase........................................................................................................... 10 1.4.3 Acetyl coenzyme-A decarbonylase synthase complex: rare organometallic reaction intermediates require unusual metal clusters .................................................. 12 Chapter 1 References ........................................................................................................ 14 CHAPTER 2 ..................................................................................................................... 16 2.1 Introduction........................................................................................................... 16 2.2 Materials and methods .......................................................................................... 20 2.2.1 Purification of Pol β and preparation of the DNA and Cr(III)·dTMPPCP substrates ....................................................................................................................... 20 2.2.2 Preparation, crystallization, and data collection of the Pol β-DNA- Cr(III)·dTMPPCP and Pol β-DNA-Cr(III)·PCP intermediate complexes..................
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