The Catalytic Mechanism of Oxalate Decarboxylase Studied by Advanced EPR Techniques

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In this award from the Chemistry of Life Processes Program in the Chemistry Division, Dr. Alexander Angerhofer, from the University of Florida, will study the molecular mechanisms by which the bacterial enzyme oxalate decarboxylase (OxDC) and specific site-directed mutants catalyze the cleavage of the carbon-carbon bond in oxalic acid. This enzyme may have important applications in the control of oxalic acid which is one of the most common naturally occurring toxins and the main ingredient in kidney stones. Advanced electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) techniques will be used to study the electronic structure and local coordination environment of two Mn ions in the protein subunit where catalysis takes place. Spin trapping will be used to investigate free radicals that are released from the protein during enzymatic turnover. Binding of substrate and various inhibitors to the enzyme will be studied with high-field EPR. These experiments will yield important kinetic, thermodynamic, and structural information about binding of substrate and inhibitors to the enzyme, and will 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. The possible existence of a long-range electron transfer chain in the protein and its role in the catalytic process will also be investigated. While OxDC is of interest in its own right and may lead to future treatments of various oxalate-related pathologies (hyperoxaluria, kidney stones, etc.), the enzyme will also serve as an example of how a protein can control reactive radical intermediates. Site-directed mutants have been generated that have decreased oxalate decarboxylase but increased oxalate oxidase activites. Work with these mutants will help to elucidate how the structure of the active site can guide the chemistry and switch between decarboxylase and oxidase activities. This project is tightly integrated into Howard Hughes Medical Institute "Science for Life" initiative at the University of Florida, which serves its undergraduate student population by bringing them into research labs early on. Each year on average two of the most promising undergraduate science majors will be directly supported in this project in the summer of their sophomore or junior years. They will have additional volunteer opportunities and perform research for credit toward their degrees which allows them to write an honors thesis during their senior year. These students will receive intensive mentoring by Dr. Angerhofer and his collaborators and will be given the opportunity to perform cutting-edge research, which will not only enrich their education but also provide them a perspective for a future career in the natural sciences. They will also have the opportunity to present their research at local, regional, and national scientific conferences. Dr. Angerhofer will make every effort to recruit students from groups that are underrepresented in the STEM disciplines.