Oxalate Decarboxylase Requires Manganese and Dioxygen for Activity

Abstract

The Bacillus subtilis oxalate decarboxylase (EC 4.1.1.2), YvrK, converts oxalate to formate and CO2. YvrK and the related hypothetical proteins YoaN and YxaG from B. subtilis have been successfully overexpressed in Escherichia coli. Recombinant YvrK and YoaN were found to be soluble enzymes with oxalate decarboxylase activity only when expressed in the presence of manga- nese salts. No enzyme activity has yet been detected for YxaG, which was expressed as a soluble protein without the requirement for manganese salts. YvrK and YoaN were found to catalyze minor side reactions: oxalate oxidation to produce H2O2; and oxalate-dependent, H2O2-independent dye oxidations. The oxalate decar- boxylase activity of purified YvrK was O2-dependent. YvrK was found to contain between 0.86 and 1.14 atoms of manganese/subunit. EPR spectroscopy showed that the metal ion was predominantly but not exclusively in the Mn(II) oxidation state. The hyperfine coupling con- stant (A ⴝ 9.5 millitesla) of the main g ⴝ 2 signal was consistent with oxygen and nitrogen ligands with hexacoordinate geometry. The structure of YvrK was modeled on the basis of homology with oxalate oxidase, canavalin, and phaseolin, and its hexameric oligomer- ization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK pos- sesses two potential active sites, only one could be fully occupied by manganese. The possibility that the C-ter- minal domain active site has no manganese bound and is buried in an intersubunit interface within the hexam- eric enzyme is discussed. A mechanism for oxalate de- carboxylation is proposed, in which both Mn(II) and O2 are cofactors that act together as a two-electron sink during catalysis.