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

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• 作者：Wen Zhu ; Lindsey M. Easthon ; Laurie A. Reinhardt ; Chingkuang Tu ; Steven E. Cohen ; David N. Silverman ; Karen N. Allen ; Nigel G. J. Richards
• 刊名：Biochemistry
• 出版年：2016
• 出版时间：April 12, 2016
• 年：2016
• 卷：55
• 期：14
• 页码：2163-2173
• 全文大小：535K
• 年卷期：0
• ISSN：1520-4995

文摘

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.