Structural and Functional Studies on Salmonella Typhimurium Ethanolamine Ammonia-Lyase Open Access
Bovell, Adonis Miguel (2013)
Abstract
Ethanolamine ammonia-lyase (EAL), a coenzyme-B12 (AdoCbl) dependent bacterial enzyme, catalyzes the deamination of select amino-alcohols by using a radical mechanism. Extensive high-resolution spectroscopic determinations of reactant intermediate-state structures and detailed kinetic and thermodynamic studies have been conducted for the Salmonella typhimurium enzyme. A statistically robust homology model for the full [(EutB-EutC)2]3 oligomer of S. typhimurium EAL is constructed from the Escherichia coli crystal structure. This structure establishes a platform for detailed, microscopic interpretation of the molecular mechanism of EAL catalysis. The model is used to describe the hierarchy of EutB and EutC subunit interactions in the native oligomer and to guide a genetic and biochemical approach to the long-standing challenge of functional oligomer reconstitution from isolated subunits. The model is used to direct site-directed mutagenesis of EAL, leading to the creation of the EutB-F258W mutant, whose fluorescence is sensitive to the binding of AdoCbl. The AdoCbl-EAL dissociation constant is determined to be 1.2 μM, which places limits on the timescale of cofactor exchange kinetics. A series of cysteine-replaced mutants of EAL was created, and progress was made towards the goal of a mutant EAL for site-directed spin labeling studies. The primary cysteine attachment site in wild-type EAL for the 4-maleimido-TEMPO spin label was identified as EutC-C37. The localization of spin labels on EAL enables the interpretation of electron paramagnetic resonance (EPR) studies that probe distal effects on protein structure caused by cofactor binding. Previously determined rate constants for decay of the cryotrapped substrate radical, and kcat values at ambient temperature, for 1H- and 2H-labelled substrate, are united in a single model that describes the sequential radical rearrangement and hydrogen atom transfer steps, from 190 to 295 K. The model indicates that hydrogen transfer proceeds via quantum mechanical tunneling, and accounts for the anomalous hydrogen isotope effects on turnover, previously observed in EAL, and other B12-dependent enzymes.
Table of Contents
1 Introduction...........................................................................................................1
1.1 Background............................................................................................................2
1.1.1 The Ethanolamine Ammonia-Lyase Reaction.............................................................4
1.1.2 Ethanolamine Utilization........................................................................................4
1.1.3 Genes Responsible for Ethanolamine Utilization........................................................5
1.1.4 Biological Significance of Ethanolamine Ammonia-Lyase.............................................7
1.1.5 Structure of Vitamin-B12........................................................................................8
1.1.6 Classes of B12-dependent enzymes........................................................................10
1.1.7 Mechanism of Action of Ethanolamine Ammonia-Lyase..............................................11
1.1.8 Previous Structural Studies of Ethanolamine Ammonia-Lyase.....................................12
1.2 Major Experimental Techniques Used in this Work.......................................................14
1.2.1 Site Directed Mutagenesis.....................................................................................14
1.2.2 Electron Paramagnetic Resonance Spin Labeling.......................................................18
1.2.2.1 Electron Paramagnetic Resonance........................................................................18
1.2.2.2 EPR Experimental Concerns.................................................................................22
1.2.2.3 Site-Directed Spin-Labeling.................................................................................23
1.2.2.3.1 Attachment of Spin Labels to Proteins................................................................23
1.2.2.3.2 Information Gleaned from Spin Labels Attached to Proteins...................................24
1.3 Overview................................................................................................................262 The Structural Model of Salmonella Typhimurium Ethanolamine Ammonia-Lyase
Directs a Rational Approach to the Assembly of the Functional [(EutB-EutC)2]3
Oligomer from Isolated Subunits...............................................................................29
2.1 Introduction...........................................................................................................30
2.2 Materials and Methods.............................................................................................33
2.2.1 Homology Modeling of S. typhimurium EAL..............................................................33
2.2.2 Protein Library.....................................................................................................36
2.2.3 Construction of the EALH6 Plasmid.........................................................................36
2.2.4 Construction of the EutBH6 and EutCH6 Plasmids.....................................................37
2.2.4.1 Preparation of the pET28a Plasmid.......................................................................37
2.2.4.2 Preparation of the EAL gene................................................................................37
2.2.4.3 Construction of the Final Plasmids.......................................................................38
2.2.5 Bacterial Growth and Purification...........................................................................38
2.2.5.1 Growth............................................................................................................38 2.2.5.2 Purification......................................................................................................392.2.5.3 Gel Filtration....................................................................................................40
2.2.6 SDS and Native PAGE..........................................................................................40
2.2.7 Enzyme Activity Assay.........................................................................................40
2.2.8 Electron Paramagnetic Resonance Spectroscopy......................................................41
2.3 Results and Discussion...........................................................................................41
2.3.1 Homology Modeling of S. typhimurium EAL.............................................................41
2.3.2 Subunit Interaction Hierarchy from the Homology Model of S. typhimurium EAL..........48
2.3.3 Expression and Purification of Wild-Type EAL..........................................................53
2.3.4 Expression and Purification of the Individual EutB and EutC Proteins..........................56
2.3.5 Characterization of the Oligomeric State of Individually Expressed and Purified EutB
and EutC....................................................................................................................57
2.3.6 Assembly of EAL from Individually Expressed and Purified EutB and EutC....................58
2.3.7 Steady-State Enzyme Activity of EAL Reconstituted from Isolated EutB and EutC..........59
2.3.8 EPR Spectroscopy of EAL Reconstituted from Isolated EutB and EutC..........................62
2.4 Conclusions...........................................................................................................643 Cobalamin Binding to Ethanolamine Ammonia-Lyase.............................................66
3.1 Introduction..........................................................................................................67
3.1.1 Background........................................................................................................67
3.1.2 Origins of Tryptophan Fluorescence.......................................................................70
3.1.3 Tryptophan Fluorescence Quenching......................................................................72
3.2 Methods...............................................................................................................75
3.3 Results and Discussion...........................................................................................76
3.3.1 Structural Considerations for Tryptophan Quenching................................................76
3.3.2 Tryptophan Fluorescence in EAL............................................................................78
3.3.3 Inner Filter Effect................................................................................................80
3.3.4 Binding Measurements on EutB.............................................................................82
3.3.5 Fluorescence Quenching of αF258W.......................................................................83
3.4 Summary.............................................................................................................864 Towards a Reactive Cysteine-Free EAL..................................................................87
4.1 Overview of Site-Directed Spin Labeling...................................................................88
4.2 Site-Directed Spin Labeling of EAL...........................................................................90
4.3 Development of a Reactive Cysteine Free EAL...........................................................92
4.3.1 Solvent Accessibility of Cysteines in EAL................................................................93
4.3.2 Single Mutant Library of EAL................................................................................94
4.3.3 Development of the Solvent-Accessible-Cysteine Free EAL mutant, EAL-5CF.............100
4.3.4 Development of EAL-5CF single mutants..............................................................103
4.3.5 Location of the Spin Labels on EAL-5CF and its Mutants.........................................111
4.3.5.1 Power Saturation Measurements......................................................................111
4.3.5.2 Mass Spectrometry Experiments......................................................................114
4.3.6 Development of the EAL-11CF Construct..............................................................121
4.3.6.1 Identification of Mutagenesis Sites....................................................................121
4.3.6.2 Production and Expression of EAL-11CF.............................................................122
4.3.6.3 Production and Expression of Separated Subunits of EAL-11CF.............................126
4.4 Insights from the Development of Cysteine Free EAL................................................130
5 Temperature-Dependence of Hydrogen Isotope Effects on the Coupled Radical
Rearrangement-Hydrogen Transfer Reaction Sequence from 190 to 295 K.............132
5.1 Introduction........................................................................................................133
5.1.1 EPR on Cryotrapped EAL....................................................................................134
5.1.2 Isotope Effects in EAL Catalysis...........................................................................135
5.1.3 Biological Hydrogen Transfer...............................................................................137
5.1.3.1 Transition State Theory...................................................................................137
5.1.3.2 Tunneling Correction to Transition State Theory..................................................140
5.1.3.3 Full Tunneling Model.......................................................................................142
5.1.3.4 Tunneling in EAL and Related Enzymes..............................................................146
5.2 Methods.............................................................................................................147
5.3 Results...............................................................................................................148
5.3.1 Aminoethanol-Generated Co(II)-Substrate Radical Pair..........................................148
5.3.2 Time-Resolved CW-EPR of Substrate Radical Decay...............................................149
5.3.3 Time and Temperature Dependence of Substrate Radical Decay..............................150
5.3.4 Steady-State Turnover of Aminoethanol...............................................................152
5.3.5 Temperature Dependence of the Isotope Effect of Substrate Radical Decay...............152
5.4 Discussion..........................................................................................................154
5.4.1 Equivalence of kcat and kobs................................................................................154
5.4.2 Comparison of 1H Reaction Kinetics from 190 to 295 K...........................................155
5.4.3 Comparison of 2H Reaction Kinetics from 190 to 295 K...........................................156
5.4.4 Kinetic Model for Substrate Radical Reaction.........................................................156
5.4.5 Temperature-Dependent Isotope Effect cannot be Explained by Activation
Entropy Differences...................................................................................................162
5.4.6 Hydrogen Transfer 2 exhibits Quantum Mechanical Tunneling..................................164
5.4.7 Estimation of the Activation Entropy Difference through the Product Radical State.....166
5.5 Conclusions........................................................................................................1676 Implications for B12 Enzymology.........................................................................169
6.1 Chapter Two: The Structural Model of Salmonella Typhimurium Ethanolamine Ammonia-Lyase Directs a Rational Approach to the Assemby of the Functional [(EutB-EutC)2]3 Oligomer...........................................................................................170 6.2 Chapter Three: Cobalamin Binding to Ethanolamine Ammonia-Lyase...........................173 6.3 Chapter Four: Towards a Reactive Cysteine-Free EAL................................................174 6.4 Chapter Five: Temperature-Dependence of Hydrogen Isotope Effects on the Coupled Radical Rearrangement-Hydrogen Transfer Reaction Sequence from 190 to 295 K..............176 6.5 Conclusions.........................................................................................................178 References..............................................................................................................179 Appendix.................................................................................................................202About this Dissertation
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