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J. Biol. Chem., Vol. 276, Issue 47, 43627-43634, November 23, 2001

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Oxalate Decarboxylase Requires Manganese and Dioxygen for Activity
OVEREXPRESSION AND CHARACTERIZATION OF BACILLUS SUBTILIS YvrK AND YoaN^*

Adam Tanner, Laura Bowater, Shirley A. Fairhurst, and Stephen Bornemann^§

From the Biological Chemistry Department, John Innes Centre, Norwich Research Park, Colney, Norwich NR4 7UH, United Kingdom

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 manganese 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 decarboxylase 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 constant (A = 9.5 millitesla) of the main g = 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 oligomerization was predicted by analogy with proglycinin and homogentisate 1,2-dioxygenase. Although YvrK possesses two potential active sites, only one could be fully occupied by manganese. The possibility that the C-terminal domain active site has no manganese bound and is buried in an intersubunit interface within the hexameric enzyme is discussed. A mechanism for oxalate decarboxylation is proposed, in which both Mn(II) and O₂ are cofactors that act together as a two-electron sink during catalysis.

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