Investigating the roles of putative active site residues in the oxalate decarboxylase from Bacillus subtilis

Abstract

Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into CO(2) and formate using a catalytic mechanism that remains poorly understood. The Bacillus subtilis enzyme is composed of two cupin domains, each of which contains Mn(II) coordinated by four conserved residues. We have measured heavy atom isotope effects for a series of Bacillus subtilis OxDC mutants in which Arg-92, Arg-270, Glu-162, and Glu-333 are conservatively substituted in an effort to define the functional roles of these residues. This strategy has the advantage that observed isotope effects report directly on OxDC molecules in which the active site manganese center(s) is (are) catalytically active. Our results support the proposal that the N-terminal Mn-binding site can mediate catalysis, and confirm the importance of Arg-92 in catalytic activity. On the other hand, substitution of Arg-270 and Glu-333 affects both Mn(II) incorporation and the ability of Mn to bind to the OxDC mutants, thereby precluding any definitive assessment of whether the metal center in the C-terminal domain can also mediate catalysis. New evidence for the importance of Glu-162 in controlling metal reactivity has been provided by the unexpected observation that the E162Q OxDC mutant exhibits a significantly increased oxalate oxidase and a concomitant reduction in decarboxylase activities relative to wild type OxDC. Hence the reaction specificity of a catalytically active Mn center in OxDC can be perturbed by relatively small changes in local protein environment, in agreement with a proposal based on prior computational studies.

Differential Substrate Specificity and Kinetic Behavior of Escherichia coli YfdW and Oxalobacter formigenes Formyl Coenzyme A Transferase

“…The extent to which active-site residues must be modified in order to change the substrate specificities of enzymes remains an interesting problem in enzyme evolution (23,44,55), and its resolution has important implications for efforts to redesign biological catalysts for biotechnological applications (11,31). The active sites of FRC and YfdW, however, are composed of conserved residues, making it difficult to understand (i) the observed differences in substrate specificity and (ii) the ability of oxalate to exhibit substrate inhibition only in the case of YfdW.…”

Observation of superoxide production during catalysis of Bacillus subtilis oxalate decarboxylase at pH 4

“…OxDC has garned attention due to its potentially numerous applications ranging from remediation of oxalate scaling in the wood and paper industry [13–16], bioengineering of crop plants for fungal resistance and lower oxalate content [17, 18], diagnostics and sensing of oxalate [19–21], bioengineering of probiotic gut bacteria to release OxDC in the intestine [22], and as a dietary supplement for the degradation of excess oxalate in the stomach [23, 24]. Despite these efforts, significant questions remain about the details of the enzymatic mechanism of OxDC, and in particular about how modifications of the enzyme can direct its chemistry away from decarboxylase to oxidase activities [25]. …”

“…This mechanism requires oxygen to cycle through its 1-electron reduced state as superoxide which would likely be protonated in the pH range in which the enzyme is active [38]. Alternative proposals suggest one of the Mn ions undergoing a redox cycle between its +2 and +3 oxidation states allowing the associated dioxygen to remain a hydroperoxyl radical throughout the reaction [25, 27, 39]. …”

“…Site-directed mutagenesis of this lid region can transform OxDC into an oxidase [41]. For decarboxylation to occur, the presence of a protonating group, tentatively assigned to be glutamate-162 in OxDC, appears to be necessary [25, 41]. The lack of such a group in OxOx may lead to a peroxycarbonate intermediate, which decays under acidic conditions to form hydrogen peroxide and carbon dioxide [27, 28, 41, 42].…”

Modeling the Metal Binding Site in Cupin Proteins

“…Second, a molecule of formate is coordinated to the N-terminal metal ion in one of the OxDC crystal structures (Anand et al, 2002). Third, recent work using OxDC mutants has shown that (i) site-directed mutagenesis of Glu162 (Svedružic et al, 2007) and (ii) modification of the N-terminal active site lid ) abolish decarboxylase activity. Although there is general agreement that Mn II in the N-terminal domain mediates OxDC-catalyzed decarboxylation, legitimate questions have been raised concerning the function (if any) of the Mn II bound in the C-terminal cupin domain.…”