Protein and Solvent Dynamical Contributions to Reactions of the 2-Aminopropanol Substrate Radical in Ethanolamine Ammonia-Lyase Pubblico

Abstract

The B12-dependent ethanolamine ammonia lyase (EAL) is the signature enzyme of the ethanolamine utilization (Eut) pathway in pathogenic strains of Salmonella and Escherichia coli associated with microbiome disease conditions in the human gut. The enzyme catalyzes the deamination of ethanolamine or the unnatural substrate, 2-aminopropanol, for production of nutrients for the bacterial cell. Under pathogenic conditions the enzyme is thought to function in the sub-cellular organelle Eut bacterial microcompartment together with the other enzymes of the Eut pathway. EAL isolated from S. typhimurium has been studied by electron paramagnetic resonance (EPR) spectroscopy to gain insights into the fundamental aspects of its molecular mechanism and modulation by the surrounding environment. The EPR spin probe, TEMPOL, identifies concentric phases around EAL: a protein associated domain (PAD, hydration layer) and an aqueous 2-aminopropanol mesodomain. The PAD undergoes the established disorder-to-order transition (ODT) with decreasing T over 230-235 K, and this transition can be tuned (T decreased) by adding (2.0% v/v) dimethylsulfoxide (DMSO) to 210-215 K. The T-dependence of the EPR amplitudes of EAL-bound paramagnets [cob(II)alamin and substrate radical] show a kink at the T values that correlates to the T of the TEMPOL-detected ODT. The kink is proposed to originate from a change in sample dielectric properties at the ODT. The kinetics of the cryotrapped cob(II)alamin-2-aminopropanol substrate radical pair decay upon temperature (T) -step initiation in the range of 210 – 240 K is measured by time-resolved, full-spectrum EPR and show parallel native and non-native (destructive) radical rearrangement pathways. The steric effect of the 2-methyl group on the substrate contributes to an increase in the barrier for the protein configurational interconversion between substrate radical states, S1^• and S2^•, that adds to the native protein configurational barrier. Simulation of the interconversion and reaction microscopic rate constants reveals that the barrier to protein configurational interconversion is raised at the T corresponding to the ODT. The results reveal the solvent and protein dynamical coupling that drives the critical protein configurational transition that bridges radical pair capture and rearrangement enabling phases of enzyme catalysis in EAL.

Table of Contents

Chapter I: Studies of EAL and Eut proteins by using EPR spectroscopy

1.1. Introduction. 2

1.1.1. The coenzyme B12: AdoCbl 5

1.1.2. Ethanolamine Ammonia Lyase: an AdoCbl-Dependent Enzyme. 7

1.1.2.1. Structure. 7

1.1.2.2. Metabolic Pathway. 8

1.1.2.3. Minimal Mechanism for EAL Catalytic Cycle and the Active Site. 10

1.1.2.4. Catalysis and Dynamics. 12

1.1.3. Bacterial Microcompartments. 14

1.1.3.1. The BMC structural protein EutS. 16

1.1.3.2. The BMC structural protein EutL. 17

1.2. Experimental techniques. 18

1.2.1. Continuous Wave EPR spectroscopy. 18

1.2.1.1. Paramagnetic molecules: TEMPOL and 4-Maleimido-TEMPO.. 21

1.2.1.2. Paramagnetic species in EAL, EPR spectroscopy. 24

1.2.1.3. Spin-Lattice Relaxation. 27

1.2.2. Site Directed Spin Labeling. 27

1.3. Outline. 29

Chapter II: Solvent dynamics around EAL in 10 mM 2-aminopropanol with 0 % and 2 % DMSO

2.1. Introduction. 31

2.2. Materials and Methods. 33

2.2.1. Protein and EPR sample preparation. 33

2.2.2. Continuous Wave EPR spectroscopy. 34

2.2.3. EPR Simulations. 35

2.3. Results. 36

2.3.1. TEMPOL EPR line shape. Temperature dependence in frozen aqueous solution with EAL and 2-aminopropanol in the presence of 0 % v/v DMSO.. 36

2.3.2. TEMPOL EPR line shape. Temperature dependence in frozen aqueous solution with EAL and 2-aminopropanol in the presence of 2 % v/v DMSO.. 36

2.3.3. TEMPOL rotational correlation times and normalized weights. Temperature dependence in frozen aqueous solution with EAL and 2-aminopropanol in the presence of 0 % v/v DMSO.. 39

2.3.4. TEMPOL rotational correlation times and normalized weights. Temperature dependence in frozen aqueous solution with EAL and 2-aminopropanol in the presence of 2 % v/v DMSO.. 41

2.4. Discussion. 43

2.4.1. 10 mM 2-aminopropanol is creates a mesodomain around EAL at cryogenic temperatures 43

2.4.2. Addition of 2 % v/v DMSO to the 10 mM 2-aminopropanol increases the mesodomain volume. 44

2.4.3. Estimations for the relative dimensions of the mesodomain. 48

2.5. Conclusions. 49

Chapter III: Mechanism of 2-aminopropanol substrate radical rearrangement catalysis in B12-dependent EAL

3.1. Introduction. 51

3.2. Materials and Methods. 53

3.2.1. Protein and EPR sample preparation. 53

3.2.2. Continuous Wave-EPR spectroscopy. 54

3.2.3. Time-resolved full-spectrum EPR spectroscopy of substrate radical decay at cryogenic temperatures. 55

3.2.4. Steady-State Kinetic Measurements. 57

3.2.5. Transient kinetics analysis. 58

3.2.6. Temperature dependence of observed rate constants. 58

3.2.7. Numerical simulations and fitting of the minimum kinetic model 59

3.3. Results. 60

3.3.1. Time-resolved, full-spectrum EPR of the cryotrapped 2-aminopropanol substrate radical intermediate in EAL. 60

3.3.2. EPR spectra of the deconvoluted Cbl(II)-radical pair species. 63

3.3.3. Quantification of the radical species contribution to the EPR spectra. Proportions of the diamagnetic product states. 64

3.3.4. Time-dependence of the Cbl(II)-substrate radical pair decay component 66

3.3.4.1. Cryotrapped 2-aminopropanol substrate radical intermediate in EAL with 0 % v/v DMSO solution, measured over 210-240 K.. 66

3.3.4.2. Cryotrapped 2-aminopropanol substrate radical intermediate in EAL with 2 and 4 % v/v DMSO solution, measured at 230 K and 240 K.. 68

3.3.5. Time-dependence of the growth of the uncoupled Cbl(II) 70

3.3.5.1. EPR measurements for EAL and 2-aminopropanol in 0 % v/v DMSO solution at T in the range 210-240 K.. 70

3.3.5.2. EPR measurements for EAL and 2-aminopropanol in 2 and 4 % v/v DMSO solution at T of 230 K and 240 K.. 72

3.3.6. Steady-state kinetic measurement 74

3.4. Discussion. 78

3.4.1. Kinetic Model. Microscopic rate constants for protein configurational change and rearrangement reaction steps. 78

3.4.2. Arrhenius behavior of the observed rate constants. 81

3.4.3. Contributions of the protein-solvent dynamical coupling to protein configurational change and rearrangement reaction steps. 85

3.5. Conclusions. 88

Chapter IV: Temperature-Dependence of the EPR Amplitude of Uncoupled Cbl(II) and Substrate Radical in EAL Provides a Direct Probe of Solvent Dynamics

4.1. Introduction. 91

4.2. Materials and methods. 92

4.2.1. Sample preparation. 92

4.2.2. Continuous wave-EPR Spectroscopy. 93

4.2.3. Progressive power saturation analysis. 94

4.3. Results and Discussion. 95

4.3.1. Paramagnetic species in EAL after reaction with 2-aminopropanol 95

4.3.2. Cbl(II)-substrate radical pair EPR line shape. Temperature dependence in frozen aqueous solution after EAL reaction with 2-aminopropanol in the presence of DMSO: 0, 2 and 4 % v/v. 98

4.3.3. Temperature dependence of the EPR amplitudes of residual and terminal species in EAL after reaction with 2-aminopropanol 99

4.3.4. Progressive Power Saturation. 102

4.4. Conclusions. 104

Chapter V: Preliminary Studies Towards Structural Investigation in Bacterial Microcompartment Proteins EutL and EutS by Electron Paramagnetic Resonance Spectroscopy

5.1. Introduction. 108

5.2. Materials and methods. 111

5.2.1. Protein cloning and expression. 111

5.2.2. Site directed mutagenesis. 112

5.2.3. Protein purification. 113

5.2.4. Fast protein liquid chromatography (FPLC) 114

5.2.5. SDS and native PAGE.. 114

5.2.6. SDSL. 115

5.2.7. CW-EPR spectroscopy. 117

5.2.8. CW-EPR simulations. 118

5.3. Results and Discussion. 118

5.3.1. Structure and sequence analysis to identify SDSL sites. 118

5.3.2. Purified Protein oligomers. 124

5.3.3. Predicted nitroxide rotamers at Cys residues in EutL and EutS. 126

5.3.4. SDSL CW-EPR of EutS. 130

5.3.5. SDSL CW-EPR of EutL A73C C201S. 133

5.4. Summary. 135

Chapter VI: Final conclusions

6.1. Summary. 138

6.2. Solvent-protein-reaction coupling in EAL.. 139

Bibliography. 142

Appendix. 150

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School	Laney Graduate School
Department	Physics
Degree	Ph.D.
Submission	Dissertation
Language	English
Research Field	Biophysics, General Physics, Molecular
Parola chiave	enzyme solvent dynamics
Committee Chair / Thesis Advisor	Warcke, Kurt, Emory University
Committee Members	Roth, Connie, Emory University Lynn, David, Emory University Finzi, Laura, Emory University Weissman, Daniel, Emory University

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