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Multidisciplinary Assessments of the Structure and Function of Co-enzyme B12-Dependent Enzyme Ethanolamine Ammonia-lyase

Sun, Li (2009)
Dissertation (183 pages)
Committee Chair / Thesis Adviser: Warncke, Kurt
Committee Members: Family, Fereydoon ; Huynh, B H Vincent ; Lynn, David ; Nagy, James
Research Fields: Chemistry, Physical; Biology, Bioinformatics
Keywords: Comparative modelling; secondary structure prediction; threading; coenzyme B12; TIM-barrel Fold; adenosylcobalamin; ethanolamine ammonia-lyase; ethanolamine deaminase; ESEEM; EPR; Optimization; Simulation; Site-directed mutagenesis
Program: Laney Graduate School, Physics
Permanent url: http://pid.emory.edu/ark:/25593/1fzqq

Abstract

The structure of the EutB protein from Salmonella typhimurium, which contains the active site of the coenzyme B12 (adenosylcobalamin)-dependent enzyme, ethanolamine ammonia-lyase (EAL), has been predicted by using comparative modeling. Multiple three-dimensional template matching servers identified predominantly β8α8, TIM-barrel proteins as templates. A conserved R160 in the active site was predicted to play a critical role in protein structure and catalysis. The protein chemical, kinetic, and electron paramagnetic resonance (EPR) and electron spin echo envelope modulation (ESEEM) spectroscopic properties of EAL with site-directed mutations in R160 have been characterized. R160I and R160E mutants fail to assemble into an EAL oligomer. The R160K and R160A mutants assemble, but R160A EAL is catalytically inactive. Simulations of the EPR spectra show that the CoII-substrate radical pair separation distances are increased by 2.1 ±1.0 Å in R160K EAL relative to wild type, which corresponds to the predicted 1.6 Å change in arginine versus lysine side chain length. 14N ESEEM from a hyperfine coupled protein nitrogen in wild type is absent in R160K EAL, which suggests that R160 interacts directly with the substrate radicals. ESEEM of the 2H-labeled substrate radical states in wild type and R160K EAL shows that the native separation distances among the substrate C1and C2, and coenzyme C5' reactant centers is conserved in the mutant protein. A ESEEM simulation toolbox, OPTESIM, was developed to facilitate the ESEEM analysis. OPTESIM allows automated numerical simulation of powder two- and three-pulse ESEEM for arbitrary number and type ( I, gN) of coupled nuclei. The EPR and ESEEM measurements evince a protein mediated force on the C5'-methyl center that is directed towards the reacting substrate species during the hydrogen atom transfer and radical rearrangement reactions. The results indicate that the positive charge at R160 side chain terminus is required for proper folding of EutB, assembly of a stable EAL oligomer, and that it is critical for catalysis. A consistent interpretation of the results is achieved by using the comparative model of EutB, which provides strong support for the model and the methodology of using this multidisciplinary approach in enzymological study.

Table of Contents


Table of Contents

Chapter 1 Introduction ......................................................................................1
§1.1 Coenzyme B12 dependent enzyme, ethanolamine ammonia-lyase ...........................2
§1.2 Previous spectroscopic study on ethanolamine ammonia-lyase ..............................5
§1.2.1 Continuous-wave EPR studies on EAL ............................................................5
§1.2.2 Electron spin echo envelope modulation (ESEEM) studies on EAL ........................8
§1.2.3 Electron-Nuclear Double Resonance (ENDOR) studies on EAL .............................14

Chapter 2 Bioinformatics study of EAL by comparative modeling of
EutB structure .................................................................................................19
§2.1 Background and introduction ................................................................ .........20
§2.1.1 Co-enzyme B12-dependent enzyme family protein structures .............................20
§2.1.2 Comparative modeling ................................................................................21
§2.2 Comparative modeling of EutB ........................................................................26
§2.2.1 Structural relationship of EutB protein to diol dehydratase and glycerol
dehydratase ......................................................................................................26
§2.2.2 Comparison with X-ray structure and model improvement ..................................39
§2.3 Predicted EutB structural features ...................................................................42
§2.3.1 N-Terminal Region of the β-Barrel: Cap Structure ............................................43
§2.3.2 Features of the Active Site .........................................................................44
§2.3.3 C-Terminal Region of the β-Barrel: Cobalamin Binding Site .................................48

Chapter 3 Comprehensive spectroscopic, and Biochemical studies on
the EAL mutants ...............................................................................................53
§3.1 Background and introduction ..........................................................................54
§3.2 Construction of a site-directed mutagenesis system and biochemical assessments
of the EAL mutants .............................................................................................56
§3.3 Spectroscopic assessments by EPR and ESEEM techniques ..................................62

§3.3.1 Theory and protocols .................................................................................62
§3.3.2 Results and discussions ..............................................................................66
§3.4 Conclusions ................................................................................................82

Chapter 4 Numerical simulation and nuclear parameter optimization of
ESEEM with OPTESIM toolbox ............................................................................86
§ 4.1 Motivation of OPTESIM toolbox .....................................................................87
§ 4.2 Simulation of ESEEM....................................................................................90
§ 4.2.1 ESEEM Theory ........................................................................................90
§ 4.2.2 OPTESIM toolbox ....................................................................................98
§ 4.3 Applications of OPTESIM .............................................................................104
§ 4.3.1 Global simulation of ESEEM from a single 14N nucleus in an enzyme
radical reaction intermediate ...............................................................................104
§ 4.3.2 Mutual orientation of two 14N hf PAS in Cu(II)-bis-histamine complex ...............108

References .....................................................................................................113

Appendices ....................................................................................................124
Appendix A. Protocol of cell growth and harvest .....................................................125
Appendix B. Protocol of EAL site-directed mutagenesis .............................................129
Appendix C. Source code of OPTESIM toolbox .........................................................131

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