The Catalytic Mechanism of Oxalate Decarboxylase Studied by Advanced EPR Experiments

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This research award in the Inorganic, Bioinorganic and Organometallic Chemistry program supports work by Professor Alexander Angerhofer at the University of Florida to study the molecular mechanism by which the bacterial enzyme oxalate decarboxylase (OxDC) catalyzes the cleavage of the carbon-carbon bond in oxalic acid. Its presence in gut bacteria helps prevent the formation of kidney stones. Advanced electron paramagnetic resonance (EPR) techniques will be used in this project to study the electronic structure and local coordination environment of its two Mn-ions in the protein subunit. A multi-frequency approach using high magnetic fields of up to 25 Tesla will be used to identify the active site. These experiments will yield important kinetic, thermodynamic, and structural information about substrate and inhibitor binding to the enzyme and test the current working hypothesis in which Mn and an associated bound oxygen molecule act as redox shuttles in the breaking of the oxalate C-C bond. Freeze-quench experiments will be employed to investigate the pre-equilibrium kinetics of enzyme-substrate interaction and yield time-dependent snapshots of the catalytic process. While the investigation of OxDC is of interest in its own right and may lead to future treatments of various oxalate-related pathologies (hyperoxaluria), the enzyme will also serve as an example of how the protein can modulate and affect catalytic reaction mechanisms. Site-directed mutants have been generated that have decreased oxalate decarboxylase but increased oxalate oxidase activity. Work with these mutants will help to elucidate how the structure of the active site can guide the chemistry. This project is tightly integrated into the HHMI-funded Science for Life initiative at the University of Florida which serves its undergraduate population by bringing them into the research lab early on. At least five of the most promising undergraduate science majors are expected to be involved in this project early in their years of study. They will receive intensive mentoring by Dr. Angerhofer and his collaborators and be given the opportunity to perform cutting-edge research which will not only enrich their education but also provide for them a perspective for a future career in the natural sciences.