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The Structure and Function Study of Three Metalloenzymes That Utilize Three Histidines As Metal Ligands

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The Structure and Function Study of Three Metalloenzymes That Utilize Three Histidines As Metal Ligands Georgia State University ScholarWorks @ Georgia State University Chemistry Dissertations Department of Chemistry Fall 11-19-2013 The Structure and Function Study of Three Metalloenzymes That Utilize Three Histidines as Metal Ligands Yan Chen Follow this and additional works at: https://scholarworks.gsu.edu/chemistry_diss Recommended Citation Chen, Yan, "The Structure and Function Study of Three Metalloenzymes That Utilize Three Histidines as Metal Ligands." Dissertation, Georgia State University, 2013. https://scholarworks.gsu.edu/chemistry_diss/86 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]. THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES THAT UTILIZE THREE HISTIDINES AS METAL LIGANDS by Yan Chen Under the Direction of Dr. Aimin Liu ABSTRACT The function of the metalloenzymes is mainly determined by four structural features: the metal core, the metal binding motif, the second sphere residues in the active site and the electron- ic statistics. Cysteamine dioxygenase (ADO) and cysteine dioxygenase (CDO) are the only known enzymes that oxidize free thiol containing molecules in mammals by inserting of a dioxygen molecue. Both ADO and CDO are known as non-heme iron dependent enzymes with 3-His metal binding motif. However, the mechanistic understanding of both enzymes is obscure. The understanding of the mechanistic features of the two thiol dioxygenases is approached through spectroscopic and metal substitution in this dissertation. Another focus of the disserta- tion is the understanding of the function of a second sphere residue His228 in a 3-His-1-carboxyl zinc binding decarboxylase α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD). ACMSD catalyzes the decarboxylation through a hydrolase-like mechanism that is initialized by the deprotonation of metal bounded water molecule. Our study reveled that the se- cond sphere residue His228 is responsible for the water deprotonation through hydrogen bond- ing. The spectroscopic and crystallographic data showed the H228Y mutation binds ferric iron instead of native zinc metal and the active site water is replaced by the Tyr228 residue ligation. Thus, we concluded that, H228Y not only plays a role of stabilizing and deprotonating the active site water but also is an essential residue on metal selectivity. INDEX WORDS: Cysteamine dioxygenase, Cysteine dioxygenase, ACMSD, Metal binding mo- tif, Metal selectivity, Substrate specificity THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES THAT UNLILZE THREE HISTIDINES AS METAL LIGANDS by Yan Chen 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 2013 Copyright by Yan Chen 2013 THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES THAT UNLILZE THREE HISTIDINES AS METAL LIGANDS by Yan Chen Committee Chair: Dr. Aimin Liu Committee: Dr. Jenny J. Yang Dr. Binghe Wang Electronic Version Approved: Office of Graduate Studies College of Arts and Sciences Georgia State University December 2013 iv DEDICATION I dedicate my dissertation to my deeply loved parents Chen, Weimin and Wang, Yaofeng. v ACKNOWLEDGEMENTS The result obtained in the dissertation is a team work of a lot of great scientists. I appreci- ate my supervisor Dr. Aimin Liu for giving me the opportunity to accomplish my research in his laboratory. I was majored in biology technology in my undergraduate school then switched to plan physiology in the graduate program in Lanzhou University. I wasn’t aware of protein purifi- cation, EPR or protein crystallization when I came to GSU in 2008. It was Dr. Liu who had spent every Saturday morning in 2008 to teach me the knowledge and the skills that needed for my Ph.D. research. I appreciate his support and training very much and I will never forget the pro- tein purification, EPR skills and the experimental tricks that he has taught me hand by hand. I would like to thank Dr. Jenny J.Yang who is always passionate and positive when she talks to me. I will always be encouraged by her positive attitude. I would like to thank Dr. Da- vidson who is a great scientist with strong scientific logics. I have learned a lot from the collabo- ration with him on MauG project. I also want to give my big thanks to Dr. Binghe Wang. Dr. Wang helped me scientifically as being my committee member. Dr. Wang also gave me unfor- gettable suggestions in my Ph.D. career. I have encountered many people in the past 5 years. Never will I forget Dr. Rong Fu, Dr. Sunil Naik, Dr. Lirong Chen and all my colleagues for their generous help during my Ph.D. study. In the past five years, I have learned the most that I can for my future. No matter how time flies, the good things and memories in Dr. Aimin Liu’s laboratory will never fade. vi TABLE OF CONTENTS THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES THAT UTILIZE THREE HISTIDINES AS METAL LIGANDS ............................................ i THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES THAT UNLILZE THREE HISTIDINES AS METAL LIGANDS ......................................... iii THE STRUCTURAL AND FUNCTION STUDY OF THREE METALLOENZYMES THAT UNLILZE THREE HISTIDINES AS METAL LIGANDS .......................................... v ACKNOWLEDGEMENTS ......................................................................................................... v LIST OF TABLES ........................................................................................................................ x LIST OF FIGURES ..................................................................................................................... xi 1 INTRODUCTION.................................................................................................................. 1 1.1 Enzymes with one histidine metal ligand ........................................................................ 2 1.2 Enzymes with two histidine metal ligands ...................................................................... 3 1.2.1 Ring cleavage dioxygenases—Extradiol and Intradiol dioxygenases ............................. 4 1.2.2 Fe/ α- ketoglutarate( αKG) dependent enzymes ................................................................. 9 1.2.3 Antibiotic biosynthesis related enzyme- Isopenicillin N synthase .................................. 12 1.3 Enzymes with three histidine metal ligands ................................................................. 14 1.3.1 Dioxygenases with 3-His-1-carboxylate motif in cupin superfamily ............................. 14 1.3.2 Dioxygenase with unique 3-His iron binding motif in cupin superfamily .................... 18 1.3.3 3-His-1-carboxylate containing enzymes amidohydrolase superfamily ........................ 22 1.3.4 Α-amino-β-carboxymuconate-ε-semialdehyde decarboxylase (ACMSD) ..................... 26 vii 1.3.5 Superoxide dismutase with 3-His-1carboxylate motif .................................................... 28 1.4 Enzymes with four histidine metal ligands ................................................................... 30 1.4.1 Cu/Zn binuclear SOD ...................................................................................................... 30 1.4.2 Superoxide reductase ....................................................................................................... 31 1.5 The overview of the dissertation .................................................................................... 34 2 MATERIAL AND METHODS ..................................................................................... 36 2.1 ADO and CDO ................................................................................................................ 36 2.1.1 The over-expression and purification of ADO................................................................ 36 2.1.2 Mössbauer spectroscopy study of ADO. .......................................................................... 37 2.1.3 EPR spectroscopy of ADO ............................................................................................... 38 2.1.4 ADO ESI mass spectrometer sample preparation .......................................................... 38 2.1.5 ADO crystallization .......................................................................................................... 39 2.1.6 Inhibition of the cysteine and homocysteine to ADO ..................................................... 40 2.1.7 DTNB treatment on ADO ................................................................................................ 41 2.2 Crystallization condition of the Fe, Co-ACMSD ......................................................... 41 3 SPECTROSPCOPIC, STRUCTURAL ANS MECHANISMTIC CHARATERIZATION OF THE THIOL DIOXYGENASES ................................... 43 3.1 EPR and Mössbauer characterization of cysteamine dioxygenase ............................ 43 3.1.1 Introduction ...................................................................................................................... 44 3.1.2 Materials and methods ..................................................................................................... 46 viii 3.1.3 Result 49 3.1.4 Discussion 57 3.2 The structure characterization
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