A Closed Conformation of Bacillus subtilis Oxalate Decarboxylase OxdC Provides Evidence for the True Identity of the Active Site

Abstract

Oxalate decarboxylase (EC 4.1.1.2) catalyzes the conversion of oxalate to formate and carbon dioxide and utilizes dioxygen as a cofactor. By contrast, the evolutionarily related oxalate oxidase (EC 1.2.3.4) converts oxalate and dioxygen to carbon dioxide and hydrogen peroxide. Divergent free radical catalytic mechanisms have been proposed for these enzymes that involve the requirement of an active site proton donor in the decarboxylase but not the oxidase reaction. The oxidase possesses only one domain and manganese binding site per subunit, while the decarboxylase has two domains and two manganese sites per subunit. A structure of the decarboxylase together with a limited mutagenesis study has recently been interpreted as evidence that the C-terminal domain manganese binding site (site 2) is the catalytic site and that Glu-333 is the crucial proton donor (Anand, R., Dorrestein, P. C., Kinsland, C., Begley, T. P., and Ealick, S. E. (2002) Biochemistry 41, 7659 -7669).The N-terminal binding site (site 1) of this structure is solvent-exposed (open) and lacks a suitable proton donor for the decarboxylase reaction. We report a new structure of the decarboxylase that shows a loop containing a 3 10 helix near site 1 in an alternative conformation. This loop adopts a "closed" conformation forming a lid covering the entrance to site 1. This conformational change brings Glu-162 close to the manganese ion, making it a new candidate for the crucial proton donor. Sitedirected mutagenesis of equivalent residues in each domain provides evidence that Glu-162 performs this vital role and that the N-terminal domain is either the sole or the dominant catalytically active domain.

Cloning and Sequencing of Two Ceriporiopsis subvermispora Bicupin Oxalate Oxidase Allelic Isoforms: Implications for the Reaction Specificity of Oxalate Oxidases and Decarboxylases

“…Homology models of the two fungal oxidase isoforms were developed, based on the structures of the bacterial decarboxylase (2,14), to help identify amino acids responsible for defining reaction specificity. The only amino acid substitutions in the N-terminal domain metal ion binding sites (site 1) of the oxidases were in an active-site lid covering the substrate binding pocket (E162S in OxoX-C and E162A in OxoX-G and T165Q in both isoforms [decarboxylase numbering]) together with a conservative change at the back of the pocket (I142L for both).…”

“…We have proposed divergent mechanisms for oxalate oxidation and oxalate decarboxylation that involve common free-radical intermediates based on the presence of Mn 2ϩ in the resting state of both enzymes and the unique requirement of dioxygen for the decarboxylase reaction (30,37). The detection of formyl radicals produced by the recombinant plant oxidase in the presence of oxalate and the crystal structures of the plant oxidase and the bacterial decarboxylase have provided additional support for such mechanisms (2,14,43,44). Recently, a detailed kinetic isotope effect study has provided the first evidence for a freeradical mechanism in the oxalate decarboxylase reaction (29).…”

Structure and Metal Loading of a Soluble Periplasm Cuproprotein

“…More than one crystal conformer of CucA was observed (supplemental Fig. 1), with some analogy to the open and closed forms of oxalate decarboxylase (32,33). Both open and closed conformations of Cu 2ϩ -CucA have been detected.…”

Modeling the Metal Binding Site in Cupin Proteins

“…This transformation is chemically interesting because the CC bond in the substrate is relatively unreactive (Begley & Ealick, 2004;Svedružic et al, 2005). X-ray crystal structures of B. subtilis OxDC and several site-specific mutants (Anand et al, 2002;Just et al, 2007;Just et al, 2004) indicate that the enzyme is a bicupin. Evidence shows that recombinant, wild-type (WT) B. subtilis OxDC contains Mn II when expressed in Escherichia coli (Angerhofer et al, 2007;Tanner et al, 2001).…”

“…The OxDC monomer is composed of two cupin, β-barrel domains, each of which contains a metal-binding site. (Just et al, 2004). Second, a molecule of formate is coordinated to the N-terminal metal ion in one of the OxDC crystal structures (Anand et al, 2002).…”