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In Vitro Catalytic Activity and Inhibition Study of Prnb from Burkholderia Ambifaria

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In Vitro Catalytic Activity and Inhibition Study of Prnb from Burkholderia Ambifaria Mississippi State University Scholars Junction Theses and Dissertations Theses and Dissertations 1-1-2012 In Vitro Catalytic Activity and Inhibition Study of PrnB from Burkholderia Ambifaria Qi Ge Follow this and additional works at: https://scholarsjunction.msstate.edu/td Recommended Citation Ge, Qi, "In Vitro Catalytic Activity and Inhibition Study of PrnB from Burkholderia Ambifaria" (2012). Theses and Dissertations. 2670. https://scholarsjunction.msstate.edu/td/2670 This Graduate Thesis - Open Access is brought to you for free and open access by the Theses and Dissertations at Scholars Junction. It has been accepted for inclusion in Theses and Dissertations by an authorized administrator of Scholars Junction. For more information, please contact [email protected]. Automated Template C: Created by James Nail 2011V2.01 In vitro catalytic activity and inhibition study of PrnB from Burkholderia ambifaria By Qi Ge A Thesis Submitted to the Faculty of Mississippi State University in Partial Fulfillment of the Requirements for the Degree of Master of Science in Chemistry in the Department of Chemistry Mississippi State, Mississippi August 2012 Copyright by Qi Ge 2012 In vitro catalytic activity and inhibition study of PrnB from Burkholderia ambifaria By Qi Ge Approved: _________________________________ _________________________________ Joseph P. Emerson Edwin A. Lewis Assistant Professor of Chemistry Professor of Chemistry (Director of Thesis) (Committee Member) _________________________________ _________________________________ Todd Mlsna Stephen C. Foster Associate Professor of Chemistry Associate Professor of Chemistry (Committee Member) (Graduate Coordinator) _________________________________ R. Gregory Dunaway Professor and Interim Dean College of Arts & Sciences Name: Qi Ge Date of Degree: August 11, 2012 Institution: Mississippi State University Major Field: Chemistry Major Professor: Joseph P. Emerson Title of Study: In vitro catalytic activity and inhibition study of PrnB from Burkholderia ambifaria Pages in Study: 52 Candidate for Degree of Master of Science PrnB is a heme-containing enzyme, which catalyzes the ring rearrangement reaction of 7-chlorotryptophan to produce 3-(3-Chloro-2-nitrophenyl)pyrrole. This thesis describes the initial isolation and characterization of PrnB, the second enzyme associated with the pyrrolnitrin biosynthetic pathway in Burkholderia ambifaria. Additionally, alternative peroxidase reactivity was used to study how amino-acids bind to the substrate binding pocket of PrnB. The peroxidase activity of PrnB was measured using three different peroxidase activity assays at various pH values. The peroxidase data was compared to similar studies with the classic peroxidase, Horseradish peroxidase (HRP). Generally, PrnB showed weak peroxidase reactivity. However this weak reactivity was an experimental handhold, where tryptophan and other substrate binding events can be explored using classic inhibition steady-state kinetics. The rate of 2-aminophenol oxidation by PrnB was used as a model assay to monitor how molecules such as L- tryptophan, L-alanine, indole, L-phenylalanine, and L-tyrosine interact with the PrnB active site. DEDICATION I would like to dedicate this research to my parents, my grandparents, and my fiancée. ii ACKNOWLEDGEMENTS I want to express my sincere appreciation to my advisor, Dr. Joseph Emerson, for his selfless time and efforts to guide and assist me throughout my Master’s degree program. I also want to say thank you to other members of my thesis committee, Dr. Edwin Lewis and Dr. Todd Mlsna for their valuable advice. iii TABLE OF CONTENTS DEDICATION .................................................................................................................... ii ACKNOWLEDGEMENTS ............................................................................................... iii LIST OF TABLES ............................................................................................................. vi LIST OF FIGURES .......................................................................................................... vii CHAPTER I. INTRODUCTION .............................................................................................1 1.1 Burkholderia ambifaria and cystic fibrosis ...........................................1 1.2 Biosynthetic pathway of pyrrolnitrin .....................................................2 1.3 Heme and heme environment ................................................................6 1.4 Heme dioxygenase .................................................................................8 II. EXPRESSION, PURIFICATION, IN VITRO CATALYTIC ASSAYS AND INHIBITION STUDY OF PRNB ..........................................................21 2.1 Expression and Purification of PrnB....................................................21 2.2 In vitro Catalytic Activity Assays ........................................................24 2.2.1 The native activity of PrnB ............................................................24 2.2.2 pH dependent o-dianisidine assay ..................................................26 2.2.3 pH dependent catechol-aniline assay .............................................27 2.2.4 pH dependent 2-aminophenol (OAP) assay ...................................28 2.2.5 Results ............................................................................................28 2.2.5.1 Result of o-dianisidine assay for both PrnB and HRP at varied pH value ........................................................................28 2.2.5.2 Result of catechol-aniline assay for both PrnB and HRP at varied pH value ....................................................................29 2.2.5.3 Result of 2-aminophenol assay for both PrnB and HRP at varied pH value ....................................................................29 2.3 Inhibition Study of PrnB Based on 2-Aminophenol Assay .................30 2.3.1 Materials and Methods ...................................................................30 2.3.2 Results ............................................................................................31 2.4 Summary ..............................................................................................33 iv REFERENCES ..................................................................................................................44 APPENDIX A. HORSERADISH PEROXIDASE (HRP) ........................................................48 v LIST OF TABLES 2.1 Buffer compositions .........................................................................................34 vi LIST OF FIGURES 1.1 Molecular structure of pyrrolnitrin ..................................................................10 1.2 Pyrrolnitrin biosynthetic pathway ....................................................................11 1.3 Gene cluster required for pyrrolnitrin biosynthesis .........................................11 1.4 Crystal structure of PrnA, PDB ID: 2JKC. ......................................................12 1.5 Generation of HOCl. ........................................................................................13 1.6 Proposed halogenation of tryptophan proceeds by electrophilic aromatic substitution at the 7 position .............................................................14 1.7 Proposed PrnD reaction mechanism ................................................................14 1.8 Crystal structure of PrnB with its native type substrate L-tryptophan, PDB ID: 2V7J. .................................................................................................15 1.9 Structure of most common hemes ....................................................................16 1.10 Crystal structure of human TDO with 4-phenylimidazole, PDB ID: 2DOT. ..............................................................................................................17 1.11 Crystal structure of X. Campestris TDO, PDB ID: 2NW7. .............................18 1.12 Two mechanic pathways for the second steps catalyzed by TDO and IDO ..................................................................................................................19 1.13 Summary of the reaction mechanisms of various heme enzymes ...................20 2.1 Result of SDS-PAGE .......................................................................................34 2.2 The linear graph of the absorption of HRP standard versus concentration ....................................................................................................35 2.3 UV-Visible absorption spectra of PrnB with varing concentration of DTT ..................................................................................................................35 2.4 In vitro native activity study of PrnB with tryptophan as substrate. ................36 vii 2.5 PrnB and HRP catalyzed the oxidation of o-dianisidine with hydrogen peroxide............................................................................................................37 2.6 PrnB and HRP catalyzed catechol-aniline assay with hydrogen peroxide............................................................................................................37 2.7 PrnB and HRP catalyzed 2-aminophenol assay with hydrogen peroxide........37 2.8 In vitro catalytic activity of oxidation of o-dianisidine by PrnB compared with HRP .........................................................................................38 2.9 In
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