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

Wen Zhu,Steven E Cohen,Nigel G J Richards,Karen N Allen,Laurie A Reinhardt,Lindsey M Easthon,David N Silverman,Chingkuang Tu

doi:10.1021/acs.biochem.6b00043

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.

Highlights

The enzymes oxalate decarboxylase (OxDC) and oxalate oxidase (OxOx) mediate the breakdown of the toxic metabolite oxalate (Scheme 1)[1] and are thought to share a common evolutionary precursor on the basis of the structural similarity and sequence identity of their “cupin” domains (Figure 1).[2,3] Even though OxDC and OxOx both require a bound manganese ion for activity,[4−6] which is coordinated by Scheme 1
Elucidating the details of the catalytic and chemical mechanisms employed by these enzymes provides an opportunity to understand how changes in protein environment might modulate metal reactivity.[7−9] it was recently reported that replacing the conformationally mobile active-site loop (Ser161-Glu162-Asn163-Ser164) in the N-terminal, Mn-binding domain of Bacillus subtilis OxDC with residues from a cognate loop (Asp161-Ala162-Ser-163-Asn164) in Ceriporiopsis subvermispora OxOx12,13 gave a variant of B. subtilis OxDC (DASN OxDC)a that exhibited considerably elevated
Hypothetical Scheme Showing How Partitioning of a Common Formyl Radical Anion Intermediate Might Lead to Different Enzyme Activities in OxDC and OxOx14,a aWe note that the metal oxidation states and dioxygen-binding site remain to be experimentally defined

Summary

Introduction

The enzymes oxalate decarboxylase (OxDC) and oxalate oxidase (OxOx) mediate the breakdown of the toxic metabolite oxalate (Scheme 1)[1] and are thought to share a common evolutionary precursor on the basis of the structural similarity and sequence identity of their “cupin” domains (Figure 1).[2,3] Even though OxDC and OxOx both require a bound manganese ion for activity,[4−6] which is coordinated by Scheme 1. Article oxidase and decreased decarboxylase activities.[14] This remarkable finding was rationalized by assuming that the first two steps in the OxDC- and OxOx-catalyzed reactions are identical and result in the formation of a Mn-bound formyl radical anion (Scheme 2), which partitions to give formate or CO2 depending on whether Glu[162] is present to protonate the carbon atom.[14] the absence of a residue (equivalent to Glu162) that is capable of mediating general acid/base catalysis in the OxOx active site[11,15] results in the production of CO2 and hydrogen peroxide, perhaps via a peroxycarbonate Scheme 2. Hypothetical Scheme Showing How Partitioning of a Common Formyl Radical Anion Intermediate Might Lead to Different Enzyme Activities in OxDC and OxOx14,a aWe note that the metal oxidation states and dioxygen-binding site remain to be experimentally defined. We report investigations on a series of OxDC active-site loop variants using X-ray crystallography, membrane inlet mass spectrometry (MIMS),[19,20] and 13(V/K) isotope effect measurements.[17,21] These studies provide the first structural insights into the mode by which oxalate coordinates the Nterminal Mn(II) ion in OxDC and suggest that the functional role of Glu[162] may be to “orient” the active-site loop into a conformation that precludes solvent access during turnover rather than to mediate the generation of a substrate-based radical intermediate as proposed previously.[16]

Methods

Results

Conclusion