| The oxalate decarboxylase from Bacillus subtilis (OxdC) catalyzes the mechanistically intriguing reaction in which the C-C bond in oxalate is cleaved to give carbon dioxide and formate. Crystallographic structures have shown that OxdC is a member of the "bicupin" structural family with a mononuclear manganese center in each cupin domain, and amino acid ligands that are structurally similar to those from oxalate oxidase, another oxalate-degrading enzyme. While oxalate oxidase catalyzes a redox reaction in which oxalate is oxidized and molecular oxygen is reduced to peroxide, OxdC catalyzes a non-redox reaction that is uniquely dependent on molecular oxygen, which is not consumed in the reaction.;In this study OxdC was overexpressed from E. coli with full manganese occupancy and a specific activity of 79 I.U./mg, equal to that obtained for the native enzyme. Oxalate 13C and 18O heavy atom isotope effects on Vmax/KM suggest the presence of a partially rate-determining step prior to the irreversible C-C bond cleavage, which was proposed to be proton-coupled electron transfer. From the pH studies it was determined that monoprotonated oxalate is the true substrate for OxdC.;Mutagenesis studies of active site arginine (R92 and 8270) and glutamate (E162 and E333) residues and heavy atom isotope effects on the reaction catalyzed by the mutants suggested that (1) the N-terminal manganese binding site hosts the catalytically active site, (2) Glu162 is the key residue determining reaction specificity and has a role of acid/base catalyst in the OxdC mechanism and (3) the role of Arg92 is to orient the oxalate molecule upon binding, polarize a C-O bond in oxalate and therefore facilitate decarboxylation.;A narrow EPR signal observed in the presence of enzyme, oxalate and dioxygen is identified as a protein-based tyrosyl radical. Kinetic studies have shown that the observed radical is not kinetically competent. Further characterization of the radical EPR signal and mutagenesis of conserved tyrosyl residues in the proximity of the Mn-binding sites suggest that the tyrosyl radical is formed more than 10 A from the Mn-binding site and in a position inaccessible to solvent. |
