CHARACTERIZATION OF HYDROLYTIC DEHALOGENASES: SUBSTRATE SPECIFICITY AND CARBON ISOTOPE FRACTIONATION A thesis submitted in conformity with the requirements for the degree of Master of Science. Graduate Department of Cell and Systems Biology University of Toronto © Copyright by Christopher Tran (2013) ABSTRACT: Characterization of Hydrolytic Dehalogenases: Substrate Specificity and Carbon Isotope Fractionation: Christopher Tran, 2013, M.Sc., Department of Cell and Systems Biology, University of Toronto The first project is focused on kinetic analysis of two enzymes: Rsc1362 (Ralstonia solanacearum GMI1000) and PA0810 (Pseudomonas aeruginosa PA01). Rsc1362 had a kcat of 504±66 min-1 and a KM of 0.06±0.02 mM, PA0810 had a kcat of 2.6±0.6 min-1 and a KM of 0.44±0.2 mM. A lack of environmental conteXt for a chloroacetate dehalogenase was noted in Pseudomonas aeruginosa PA01. The second project focuses on kinetic and stable isotope fractionation of 1,2- dichloroethane by DhlA (Xanthobacter autotrophicus GJ10), and Jann2620 (Jannaschia CCS1). Although both enzymes had different kinetics (DhlA: KM = 4.8±0.6 mM and kcat = 133±8 min-1, Jann2620: KM = 25.9±2.3 mM and kcat = ~1.7 min-1), they fractionated similarly (ε values of -33.9‰ and -32.9‰ for DhlA and Jann2620, respectively). As calculated AKIE values were similar to the eXpected values of an abiotic reaction, it was determined that neither enzyme masks the intrinsic fractionation. ii Table of Contents ABSTRACT: ................................................................................................................................ ii LIST OF TABLES: ....................................................................................................................... v LIST OF FIGURES: ................................................................................................................... vi 1.0 INTRODUCTION: ................................................................................................................ 1 1.1 Halogenated Organic Compounds ........................................................................................ 1 1.2 Hydrolytic Dehalogenation .................................................................................................... 2 1.3 Motivation and Goals ................................................................................................................ 4 1.4 Background relating to Project 1: Structure/Function relationships between Defluorination and Dechlorination Specific Enzymes ......................................................... 7 1.4.1 Recent Genomic Screen reveals Novel Defluorinating Haloacid Dehalogenases ..... 7 1.4.2 Origins of the enzymes studied: Parent Species ..................................................................... 9 1.4.3 Rationale and Breakdown of Project 1 .................................................................................... 10 1.5 Background relating to Project 2: Enzyme Efficiency and Carbon Isotope Fractionation ................................................................................................................................... 10 1.5.1 Compound Specific Isotope Analysis ........................................................................................ 10 1.5.2 Stable Isotope Fractionation ........................................................................................................ 13 1.5.3 Apparent and Intrinsic Kinetic Isotope Effects .................................................................... 15 1.5.4 Rationale and Formation of Project #2 ................................................................................... 17 1.6 Additional Background Information on Haloacid and Haloalkane dehalogenases: ............................................................................................................................... 19 1.6.1 Haloacid Dehalogenases ................................................................................................................. 19 1.6.2 Haloalkane Dehalogenases ........................................................................................................... 21 2.0 METHODS: ........................................................................................................................ 23 2.1 Recombinant Cell Lines: ....................................................................................................... 23 2.2 Recombinant Protein Purification: ................................................................................... 23 2.2.1 Protein Quantification and Quality control: .......................................................................... 24 2.3 pH Optimization Assays: ....................................................................................................... 25 2.4 pH Optima Analysis and Quantification .......................................................................... 26 2.4.1 Ion Chromatography (HAD pH optimization): ..................................................................... 26 2.4.2 Ion Chromatography (HLD pH optimization): ..................................................................... 26 2.5 Isothermal Titration Calorimetry (HAD Kinetic Analysis): ...................................... 27 2.6 Enzyme Kinetics: Isothermal Titration Calorimetry ............................................................. 28 2.7 Carbon Stable Isotope Fractionation and 1,2 DCA Kinetics of HLDs: .................... 31 3.0 PROJECT 1 RESULTS: CHARACTERIZATION OF HADs Rsc1362 AND PA0810 ..................................................................................................................................................... 33 3.1 Rsc1362 and PA0810 pH Optima ...................................................................................... 33 3.2: Isothermal Titration Calorimetry and Kinetic Analysis ........................................... 34 3.2.1 Determining Apparent Enthalpy of Reaction: ...................................................................... 34 3.2.2 Enzyme Kinetics: ............................................................................................................................... 35 3.3 Structural Data: ....................................................................................................................... 39 4.0: PROJECT 1 DISCUSSION: HALOACID DEHALOGENASE CHARACTERIZATION ..................................................................................................................................................... 43 4.1: Advantages/Disadvantages of Isothermal Titration Calorimetry in Enzyme Kinetic Analysis .............................................................................................................................. 43 iii 4.2 Kinetic and pH optima Data Suggests Chloroacetate is not the Primary Substrate of PA0810 ..................................................................................................................... 44 4.3: Environmental Background of Microbial Strains ....................................................... 46 4.4: Structural Explanations for the Difference in Activity Between Rsc1362 and PA0810 .............................................................................................................................................. 47 4.5: Conclusions for the Overall Project: Structural Adaptations for Defluorination .............................................................................................................................................................. 47 5.0 PROJECT 2 RESULTS: KINETICS AND ISOTOPE FRACTIONATION .................. 49 5.1 pH Optima of HLD Enzymes ................................................................................................. 49 5.2: Degradation of 1,2-DCA by DhlA and Jann2620 .......................................................... 51 5.3: Carbon Isotope Fractionation Data for DhlA and Jann2620 ................................... 52 5.4: Enzyme Kinetic Data ............................................................................................................. 57 5.5: Calculation of AKIE ................................................................................................................ 61 5.6: Controls ..................................................................................................................................... 62 6.0 PROJECT 2 DISCUSSION: HALOALKANE DEHALOGENASE CHARACTERIZATION ........................................................................................................... 65 6.1 General Discussion ................................................................................................................. 65 6.2 Analysis of pH Optima Data ................................................................................................. 67 6.3 Analysis of Enzyme Kinetic Data ........................................................................................ 67 6.4 Carbon Isotope Fractionation Analysis ........................................................................... 69 6.5 Final Comments on Biotic Masking of KIE ...................................................................... 72 7.0 Conclusions: ..................................................................................................................... 75 7.1 Project 1 HAD conclusions: ................................................................................................