The goal of the proposed research is to elucidate fundamental physical-chemical principles that govern catalysis in enzymes. This goal will be enacted through a program of biochemical and physical studies of representatives of all three classes of coenzyme B12 (adenosylcobalamin) -dependent enzymes: Methylmalonyl-CoA mutase (MCM, Class I; human, Methylobacter extorquens), ethanolamine ammonia-lyase (EAL, Class II; Salmonella typhimurium), and Lysine-5,6-aminomutase (Class III; LAM, Clostridium stricklandii). The inquiry will be extended to address mechanism in the intracellular cobalamin (B12) trafficking pathway in humans by the CblC protein (human, Caenorhabditis elegans). Innovative methods, software and hardware for pulsed-electron paramagnetic resonance (EPR) spectroscopy, in conjunction with biochemical techniques and developed low-temperature systems enable pioneering experiments. The proposed entropically-driven, configurational catalysis mechanism will be validated and developed for the radical generation step in the Class I, II, and III B12 enzymes. The contributions of protein and coupled solvent dynamics to radical rearrangement catalysis in EAL will be augmented by high-resolution determination of the reactant and protein structures that underlie the free energy landscape. Significant biomedical and human health outcomes include: (i) Unique, fundamental knowledge about the role of multi-configurational, high-entropy protein states in enzyme function, that contribute new tools and models to the developing roadmap for leveraging these states in enzyme engineering and drug development, (ii) Characterization of EAL, which is a central player in the role of the microbiome in disease progression, including links with inflammatory bowel disease, obesity, and diabetes, (iii) Insights into the molecular mechanistic basis of human metabolic disorders identified with the cbl gene cluster, and in particular, the role of the CblC protein (also known in humans as the MMACHC, methylmalonic aciduria type C and homocystinuria, protein).

Public Health Relevance

The goal of the proposed research is to elucidate fundamental physical-chemical principles that govern catalysis in enzymes. Biochemical and physical studies of representatives of all three classes of B12-dependent enzymes, and an intracellular cobalamin trafficking protein, enabled by innovations in EPR spectroscopic methods, software and hardware, will reveal how multi-configurational, high-entropy protein states contribute to enzyme function. Significant biomedical and human health outcomes include new tools and models for enzyme engineering and drug development, characterization of an enzyme that is a central player in the role of the human gut microbiome in disease progression, and insight into the molecular mechanistic basis of human metabolic disorders, including methylmalonic aciduria and homocystinuria.

Agency
National Institute of Health (NIH)
Institute
National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK)
Type
Research Project (R01)
Project #
5R01DK054514-17
Application #
9391187
Study Section
Special Emphasis Panel (ZRG1)
Program Officer
Sechi, Salvatore
Project Start
1998-03-15
Project End
2019-11-30
Budget Start
2017-12-01
Budget End
2018-11-30
Support Year
17
Fiscal Year
2018
Total Cost
Indirect Cost
Name
Emory University
Department
Physics
Type
Schools of Arts and Sciences
DUNS #
066469933
City
Atlanta
State
GA
Country
United States
Zip Code
30322
Kohne, Meghan; Zhu, Chen; Warncke, Kurt (2017) Two Dynamical Regimes of the Substrate Radical Rearrangement Reaction in B12-Dependent Ethanolamine Ammonia-Lyase Resolve Contributions of Native Protein Configurations and Collective Configurational Fluctuations to Catalysis. Biochemistry 56:3257-3264
Wang, Miao; Zhu, Chen; Kohne, Meghan et al. (2015) Resolution and Characterization of Chemical Steps in Enzyme Catalytic Sequences by Using Low-Temperature and Time-Resolved, Full-Spectrum EPR Spectroscopy in Fluid Cryosolvent and Frozen Solution Systems. Methods Enzymol 563:59-94
Robertson, Wesley D; Bovell, Adonis M; Warncke, Kurt (2013) Cobinamide production of hydrogen in a homogeneous aqueous photochemical system, and assembly and photoreduction in a (??)8 protein. J Biol Inorg Chem 18:701-13
Wang, Miao; Warncke, Kurt (2013) Entropic origin of cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase. J Am Chem Soc 135:15077-84
Chen, Hanlin; Sun, Li; Warncke, Kurt (2013) Heterogeneous ordered-disordered structure of the mesodomain in frozen sucrose-water solutions revealed by multiple electron paramagnetic resonance spectroscopies. Langmuir 29:4357-65
Hernández-Guzmán, Jessica; Sun, Li; Mehta, Anil K et al. (2013) Copper(II)-bis-histidine coordination structure in a fibrillar amyloid ?-peptide fragment and model complexes revealed by electron spin echo envelope modulation spectroscopy. Chembiochem 14:1762-71
Sun, Li; Savory, Joshua J; Warncke, Kurt (2013) Design and implementation of an FPGA-based timing pulse programmer for pulsed-electron paramagnetic resonance applications. Concepts Magn Reson Part B Magn Reson Eng 43:100-109
Bovell, Adonis Miguel; Warncke, Kurt (2013) The structural model of Salmonella typhimurium ethanolamine ammonia-lyase directs a rational approach to the assembly of the functional [(EutB-EutC)ýýý]ýýý oligomer from isolated subunits. Biochemistry 52:1419-28
Gunderson, William A; Hernández-Guzmán, Jessica; Karr, Jesse W et al. (2012) Local structure and global patterning of Cu2+ binding in fibrillar amyloid-? [A?(1-40)] protein. J Am Chem Soc 134:18330-7
Robertson, Wesley D; Wang, Miao; Warncke, Kurt (2011) Characterization of protein contributions to cobalt-carbon bond cleavage catalysis in adenosylcobalamin-dependent ethanolamine ammonia-lyase by using photolysis in the ternary complex. J Am Chem Soc 133:6968-77

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