This project seeks to elucidate how protein environment modulates the chemistry of oxalate degradation in recombinant oxalate oxidase from Ceriporiopsis subvermispora. Oxalate oxidase catalyzes the carbon-carbon bond cleavage of oxalate to yield carbon dioxide and hydrogen peroxide. Although there is currently no structural information available for oxalate oxidase from C. subvermispora (CsOxOx), sequence data and homology modeling indicate that it is the first manganese-containing bicupin enzyme identified that catalyzes this reaction. The best characterized oxalate oxidases are from barley and wheat. These enzymes, also known as germins, contain a single cupin domain and are therefore classified as monocupins. Interestingly, CsOxOx shares greatest sequence homology with the bicupin microbial oxalate decarboxylases. The short term goals of this research are to 1) to characterize the manganese-dependence of oxalate oxidase from C. subvermispora and 2) to identify the active site of the enzyme. This research is expected to contribute to the understanding of how subtle structural changes effect remarkable functional variation in evolutionarily related proteins. Characterizing the manganese-dependence of CsOxOx is significant in order to place this enzyme in the context of other oxalate degrading enzymes and that of other cupin proteins. Identifying the active site of CsOxOx is an important problem. If only one of the manganese centers mediates catalysis, critical questions are raised concerning the function (if any) of the second Mn-binding domain and the extent to which local protein structure in each domain results in differential reactivity.
Broader Impacts This project is transforming Gainesville State College (a two-year unit of the University System of Georgia) from an institution that provides excellent coursework in the sciences into one that also provides students the opportunity to participate in meaningful and important laboratory research. Undergraduate students carry out most of this work at Gainesville State College (GSC), but also have the opportunity to work with collaborators at the University of Florida and the National High Magnetic Field Laboratory in Tallahassee, FL. Student researchers are expected to communicate their research experiences to broad audiences through diverse media. Student researchers also are expected to publish their results and report research findings at professional meetings and to the regional educational community. Funds from this project are used to acquire, operate, and maintain research equipment that is not otherwise available at GSC.
This project integrated the excitement of laboratory-based scientific discovery into undergraduate education by providing high-quality research opportunities at the interface of chemistry, biology, and physics by addressing how protein environment modulates chemical reactivity in oxalate oxidase from Ceriporiopsis subvermispora (CsOxOx). Oxalate oxidase catalyzes the carbon-carbon bond cleavage of oxalate to yield carbon dioxide and hydrogen peroxide. Although there is currently no structural information available for CsOxOx, sequence data and homology modeling indicate that it is the first bicupin enzyme identified that catalyzes this reaction. The best characterized oxalate oxidases are from barley and wheat. These enzymes contain a single cupin domain and are therefore classified as monocupins. Interestingly, CsOxOx shares greatest sequence homology with the bicupin microbial oxalate decarboxylases. The characterization of CsOxOx has provided the field of biochemistry with an opportunity to compare and contrast this enzyme with other enzymes possessing similar structural attributes yet catalyzing very different reactions. We have shown that CsOxOx activity directly correlates with manganese content and that other metals do not support catalysis. Electron paramagnetic resonance spectra indicate that the Mn is present as Mn(II) and are consistent with the coordination environment expected from homology modeling with known X-ray crystal structures of oxalate decarboxylase from Bacillus subtilis (BsOxDC). We have shown that acetate and a number of other small molecule carboxylic acids are competitive inhibitors for oxalate in the CsOxOx catalyzed reaction. We have identified where on the enzyme catalysis takes place (the N-terminal Mn-binding site). Experiments support the existence of an oxalate-derived radical species formed during enzyme turnover. These data strongly support the formation of a carbon dioxide radical as an intermediate in the proposed catalytic mechanism. Trapping of the same species in oxalate decarboxylase is consistent with a carbon dioxide radical intermediate formed during turnover in both BsOxDC and CsOxOx. We adapted the use of membrane inlet mass spectrometry to characterize the catalytic properties of CsOxOx through the continuous, real-time direct detection of oxygen consumption and carbon dioxide production and showed that nitric oxide inhibits CsOxOx. These results, coupled with our construction of protein similarity networks and the application of computational and bioinformatics techniques, have allowed us to place the enzyme within the context of the larger cupin superfamily and to contribute to the understanding of how protein structure modulates chemical reactivity in evolutionarily related enzymes. Fifteen individuals (twelve undergraduate students, two high school students, and a high school teacher) directly participated in this NSF funded research. Of these participants seven are women, three are underrepresented minorities, and one is an international exchange student from Brazil. Their contributions have resulted in twenty one student presentations (two at international meetings, eleven at regional meetings, and seven at local meetings). Eight undergraduate researchers are authors on one or more publications. Not only were the NSF sponsored student researchers part of meaningful and important research carried out at their home institution, they also worked in collaboration with scientists at the Enzyme Function Initiative, the University of Florida, and the National High Magnetic Field Laboratory (NHMFL). The mutual communication facilitated by these collaborative undertakings enhanced the infrastructure for research and education at all of the involved institutions. The NSF sponsored student researchers involved in this project were irreversibly transformed by increasing their abilities to learn and work independently as well as collaboratively, by strengthening their oral and written communication skills, by sharpening their critical thinking skills, and by enhancing their resumes. Their research experiences were the triumphant archway into the competence and confidence required to achieve success in the sciences in the global community.
