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.
Research outcome from this award can be summarized as follows: Using EPR (electron paramagnetic resonance) combined with a fluorescent oxygen sensor, we were able to identify a key intermediate radical in the enzymatic mechanism by which oxalate decarboxylase breaks down oxalate. The radical in question is the carbon dioxide radical anion and it appears to be a signature of residual oxidase activity of the enzyme. The release of this radical from the protein is affected by a flexible section of the amino acid chain, SENST161-165. The simplest explanation of our experiments suggests that the residual oxidase activity of the enzyme is due to loss of control of the radical intermediate by the protein and that the decarboxylase activity depends on the successful conversion of the radical into product formate. We were also able to show that oxalate binds bidentate to the active site in the enzyme. This information is important because it corrects previous hypotheses about the enzyme mechanism and it suggests an alternative site for oxygen binding. Furthermore, we were able to identify at least five different Mn(II) species in oxalate decarboxylase. Moreover, we were able to develop new intensity and field standards for high-field EPR which makes this method more reliable and useful for biophysical research. These results are significant because they allow a much clearer picture of the detailed molecular mechanism by which oxalate decarboxylase breaks down oxalate, one of the most common naturally occurring toxins, into carbon dioxide and formate. The enzyme has many potential applications in medicine (potential kidney stone remedy, enzyme assay for blood oxalate concentration, etc.), the agricultural and food industries (reduction of oxalate in the food chain), and the paper/pulp industry (cleaning of process equipment from oxalate deposits), and a thorough understanding of its molecular mechanism is key to its many potential applications. Eight undergraduate students (three female) participated in this NSF-funded research project and were partially supported by it. Of these, five have moved on to advanced studies in medical or pharmacy schools in the state of Florida, while the rest are still working toward their degrees and are continuing their research in the PI's lab. Two graduate students and one postdoctoral associate were partially supported through this award. All of these individuals actively participated in research at the interface between chemistry, biology, and physics. They acquired skills in protein preparation and handling, spectroscopy (in particular EPR spectroscopy), data acquisition and analysis, and in communicating scientific ideas. All presented their research in regional conferences (Annual Meeting of the Florida Section of the ACS, and the Annual Southeastern Magnetic Resonance Conference), and some were able to attend larger national and international meetings (Annual Meeting of the Society for Free Radicals in Biology and Medicine, and the Pacifichem conference). All students and the postdoc placed into the appropriate next level of their careers after they completed their research in our lab, such as Medical or Pharmacy School for the undergraduate students and academic jobs for the graduate student and postdoctoral associate. Our lab also participated in the SSTP (Student Science Training Program) program at the University of Florida during two summers. This allowed two high school students to participate in our research during seven weeks each in the summers of 2010 and 2012. The two students presented their results in a poster session and a short oral presentation in front of their peers. The PI presented the various aspects of this research during numerous national and international conferences and in invited talks at academic institutions.
