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A Biophysical Study of Fluoroacetate Dehalogenase A Biophysical Study of Fluoroacetate Dehalogenase By Pedram Mehrabi A thesis submitted in conformity with the requirements for the degree of Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto © Copyright by Pedram Mehrabi 2017 A Biophysical Study of Fluoroacetate Dehalogenase Pedram Mehrabi Doctor of Philosophy Graduate Department of Medical Biophysics University of Toronto 2017 Abstract Fluoroacetate dehalogenase (FAcD) is a homodimeric enzyme and belongs to the α/β hydrolase protein superfamily. It possesses the ability to cleave C-F bonds and so is capable of using the simplest fluorocarbon, fluoroacetate, as its main source of carbon. The underlying mechanistic details that allow this enzyme to perform C-F cleavage are still unknown. However, details into building a mechanistic model for the type of asymmetric cooperative behavior observed in FAcD are slowly being revealed. In the following studies, I use FAcD as a model system to study its catalytic reaction mechanism focusing on the enzymatic coordinate pathway and the dynamics of various states along the pathway. As well, I use FAcD as a suitable choice to study irreversible enzymatic reactions via time-resolved crystallography (TRX) experiments. Using a series of biophysical techniques I analyze structure, function, and dynamics of both the ground state and transient higher energy states. It was demonstrated using X-ray crystallography that there is subtle structural asymmetry between the individual subunits in the apo state of the enzyme. This asymmetry becomes more pronounced during catalytic activity whereby entropic loss from substrate binding in one protomer is compensated by an increase of entropy and a loss of structural waters in the other. ii Kinetic measurements of FAcD revealed that during titrations involving excess substrate the enzyme undergoes substrate inhibition. Biphasic chemical shifts identified by NMR indicated a secondary binding site, which was confirmed via crystal structures of various complexes of a catalytically slow mutant (Y219F). This secondary binding site decreases transmission along the allosteric pathway, which could be mapped using rigidity-based transmission allostery (RTA) analysis. Using FAcD as a model system for method development I applied fixed target approaches for time-resolved crystallography (TRX). Using light activation of a caged-substrate, I could verify that structural asymmetry first observed using a series of mutants is still conserved. As well, structural features of the active site and cap domain change from one time-point to the next and corroborate previous results. Taken together, this data shows nuanced mechanistic insights into the structural enzymology of a homodimeric enzyme. iii Acknowledgements The frontiers of science are now only rarely pushed forth as the work of individuals, but rather involve collaborative efforts in the hopes of being able to understand more about the character of Nature. Several people contributed to my research which made the work of this thesis possible. I would like to start off by expressing my sincere appreciation to my supervisor, Dr. Emil Pai for offering me an opportunity to work on a project that satisfied many of my scientific curiosities and opened many new ones. Not only for his guidance, but also for consistently allowing me the opportunity to bounce my ideas off him. I would also like to express my gratitude to my committee members Drs. Scott Prosser and Gil Privé. Additional thanks goto Dr. Scott Prosser for his guidance and keen insights into understanding protein dynamics. Further thanks go to the members of the Pai lab for creating an enjoyably working environment. In particular, I would like to thank Ondrej Halgas and Natasha Krugylak for interesting conversation and for their comradery. I would like to also thank my many collaborators which made much of this research possible. Dr. Zhong Ren for introducing me to both dynamic crystallography and SVD analysis all while unwearyingly answering all my questions. Dr. Adnan Sljoka, for showing me a new world of protein analysis. Dr. Vukica Srajer for introducing me to Laue time-resolved crystallography. Drs. Henrike Müller-Werkmeister and Eike Schulz for tirelessly working through very long beamtimes, trying to optimize and improve our time-resolved measurements. As well as the many members of DESY beamtimes, especially Jessica Besaw, Antoine Sarracini, and Kamil Krawczyk. I would especially like to thank Dr. Tae Hun Kim, not only for his comradery, but also for spending countless hours with me in the lab, patiently teaching me NMR and diligently going over data. Last but not least, I would like to thank my parents Fariba and Saeid for their constant love, support and encouragement. iv Table of Contents Abstract ........................................................................................................................................ ii Acknowledgements ..................................................................................................................... iv List of Tables ............................................................................................................................... ix List of Figures .............................................................................................................................. x List of Appendices .................................................................................................................... xiv List of Abbreviations ................................................................................................................. xv Chapter 1 ...................................................................................................................................... 1 1.1 Halogenated hydrocarbon waste ..................................................................................... 1 1.1.1 Bioremediation ........................................................................................................ 1 1.1.2 Microbial Dehalogenation ....................................................................................... 3 1.1.2.2 Thiolytic dehalogenation .................................................................................. 4 1.1.2.3 Oxygenolytic dehalogenation ........................................................................... 5 1.1.2.4 Dehydrohalogenation ....................................................................................... 6 1.1.2.5 Reductive dehalogenation ................................................................................ 7 1.1.3 Hydrolytic dehalogenation....................................................................................... 8 1.1.3.1 Haloacid dehalogenases ................................................................................... 8 1.1.3.2 Haloalkane dehalogenases ................................................................................ 9 1.1.3.3 Fluoroacetate dehalogenases .......................................................................... 10 1.2 Structural approaches to study fluoroacetate dehalogenase .......................................... 12 1.2.1 Classical X-ray crystallography ............................................................................. 13 1.2.2 Time-resolved crystallography .............................................................................. 14 1.2.2.1 Ideal systems .................................................................................................. 16 1.2.2.2 Mutational Trapping ....................................................................................... 16 1.2.2.3 Physical Trapping ........................................................................................... 17 1.2.2.4 Analytical Trapping ........................................................................................ 17 v 1.2.3 Structure determination by serial crystallography: an overview ........................... 18 1.2.4 Computational approaches to electron density interpretation ............................... 20 1.2.4.1 Singular value decomposition ........................................................................ 21 1.2.4.2 Absolute value electron density maps ............................................................ 22 1.3 Allostery ........................................................................................................................ 23 1.3.1 Models of allostery ................................................................................................ 23 1.3.1.1 Concerted Model ............................................................................................ 23 1.3.1.2 Sequential Model ............................................................................................ 23 1.3.1.3 Ensemble Model ............................................................................................. 25 1.3.2 Methods to study allostery ..................................................................................... 26 1.3.2.1 X-ray crystallography ..................................................................................... 26 1.3.2.2 Nuclear Magnetic Resonance ......................................................................... 26 1.3.2.3 Computational approaches ............................................................................. 28 1.4 Thesis Rationale ...........................................................................................................
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