Substrate Binding Mode and Molecular Basis of a Specificity Switch in Oxalate Decarboxylase

Abstract

Oxalate decarboxylase (OxDC) catalyzes the conversion of oxalate into formate and carbon dioxide in a remarkable reaction that requires manganese and dioxygen. Previous studies have shown that replacing an active-site loop segment Ser161-Glu162-Asn163-Ser164 in the N-terminal domain of OxDC with the cognate residues Asp161-Ala162-Ser-163-Asn164 of an evolutionarily related, Mn-dependent oxalate oxidase gives a chimeric variant (DASN) that exhibits significantly increased oxidase activity. The mechanistic basis for this change in activity has now been investigated using membrane inlet mass spectrometry (MIMS) and isotope effect (IE) measurements. Quantitative analysis of the reaction stoichiometry as a function of oxalate concentration, as determined by MIMS, suggests that the increased oxidase activity of the DASN OxDC variant is associated with only a small fraction of the enzyme molecules in solution. In addition, IE measurements show that C–C bond cleavage in the DASN OxDC variant proceeds via the same mechanism as in the wild-type enzyme, even though the Glu162 side chain is absent. Thus, replacement of the loop residues does not modulate the chemistry of the enzyme-bound Mn(II) ion. Taken together, these results raise the possibility that the observed oxidase activity of the DASN OxDC variant arises from an increased level of access of the solvent to the active site during catalysis, implying that the functional role of Glu162 is to control loop conformation. A 2.6 Å resolution X-ray crystal structure of a complex between oxalate and the Co(II)-substituted ΔE162 OxDC variant, in which Glu162 has been deleted from the active site loop, reveals the likely mode by which the substrate coordinates the catalytically active Mn ion prior to C–C bond cleavage. The “end-on” conformation of oxalate observed in the structure is consistent with the previously published V/K IE data and provides an empty coordination site for the dioxygen ligand that is thought to mediate the formation of Mn(III) for catalysis upon substrate binding.

The Metal Drives the Chemistry: Dual Functions of Acireductone Dioxygenase

“…A similar mode of substrate binding, where oxalate binds in an end-on conformation providing an empty coordination site for dioxygen ligand was observed in oxalate decarboxylase. 69 …”

The Structure of Oxalate Decarboxylase at its Active pH

“…Yet, the presence of dioxygen is obligatory for catalysis and O2 is generally considered to act as a co-catalyst (6). Several crystal structures of the enzyme have appeared in the literature over the past 16 years all of which refer to enzyme poised at a pH of 7.5 or higher (7)(8)(9)(10)(11)(12)(13)(14). Yet, OxDC is only active at low pH with peak activity at or below pH4 and negligible activity above pH7 (15).…”

“…Using a series of lid mutants, Burrell et al concluded that E162 is essential for decarboxylase activity and suggested that it acts as the transient base that picks up the acidic proton from the oxalate monoanion during an initial PCET step (10). E162 turns inward toward the N-terminal Mn as seen in the closed-loop crystal structure by Just et al (8) However, this conformation is unlikely to be the catalytically active one since it presents a steric clash with substrate bound to Mn (13). Saylor et al reported on the structure of the T165V mutant enzyme which showed carbonate (or bicarbonate, a competitive inhibitor (20)) bound in the active site (11).…”

“…The fact that the T165V mutant exhibits a ten-fold lower decarboxylase activity compared to wild-type was interpreted as the result of this apparent 'mis-folding' of E162 into an unproductive conformation (11). More recently, Zhu et al reported on the structure of a ΔE162 deletion mutant where Mn was substituted for Co (13). The deletion mutant contains oxalate in a mono-dentate end-on orientation which fits into the space vacated by the absence of E162.…”

“…Based on modelling of the void space Zhu et al rationalized that the original closed conformation of the SENST lid is only present in the absence of substrate or other small carboxylate ligands. They further argue that E162 may not function as a transient base in shuttling a proton back and forth but rather as a gating mechanism that locks the loop in the closed conformation during catalysis in order to prevent solvent access to the intermediate radicals (13).…”