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Substrate binding mode and molecular basis of a specificity switch in oxalate decarboxylase

Zhu, Wen, Easthon, Lindsey M., Reinhardt, Laurie A., Tu, Chingkuang, Cohen, Steven E., Silverman, David N, Allen, Karen N and Richards, Nigel G. J. 2016. Substrate binding mode and molecular basis of a specificity switch in oxalate decarboxylase. Biochemistry 85 (14) , pp. 2163-2173. 10.1021/acs.biochem.6b00043

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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 by 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, however, 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 increased solvent access 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 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.

Item Type: Article
Date Type: Published Online
Status: Published
Schools: Chemistry
Subjects: Q Science > QD Chemistry
Additional Information: Publication Date (Web): March 25, 2016 Pdf uploaded in accordance with publisher's policy at http://www.sherpa.ac.uk/romeo/issn/0006-2960/
Publisher: American Chemical Society
ISSN: 0006-2960
Date of First Compliant Deposit: 31 March 2016
Date of Acceptance: 25 March 2016
Last Modified: 20 Dec 2017 20:03
URI: http://orca.cf.ac.uk/id/eprint/88326

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