Structural and Spectroscopic Studies Shed Light on the Mechanism of Oxalate Oxidase

Olaniyi Opaleye,Ruth-Sarah Rose,Mei M Whittaker,Eui-Jeon Woo,James W Whittaker,Richard W Pickersgill

doi:10.1074/jbc.m510256200

Olaniyi Opaleye, Ruth-Sarah Rose + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m510256200

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Abstract

Oxalate oxidase (EC 1.2.3.4) catalyzes the conversion of oxalate and dioxygen to hydrogen peroxide and carbon dioxide. In this study, glycolate was used as a structural analogue of oxalate to investigate substrate binding in the crystalline enzyme. The observed monodentate binding of glycolate to the active site manganese ion of oxalate oxidase is consistent with a mechanism involving C-C bond cleavage driven by superoxide anion attack on a monodentate coordinated substrate. In this mechanism, the metal serves two functions: to organize the substrates (oxalate and dioxygen) and to transiently reduce dioxygen. The observed structure further implies important roles for specific active site residues (two asparagines and one glutamine) in correctly orientating the substrates and reaction intermediates for catalysis. Combined spectroscopic, biochemical, and structural analyses of mutants confirms the importance of the asparagine residues in organizing a functional active site complex.

Highlights

Oxalate oxidase is widespread in nature and has been found in bacteria [4], fungi [1, 5], and various plant tissues [6]
The need to establish the details of the catalytic mechanism of Oxalate oxidase (OXO) and the importance of doing so for understanding the evolution of biological catalysis was highlighted in a recent review [15]
In the present work we describe the crystal structure of a substrate-analogue binary complex that allows, for the first time, identification of residues that bind substrates and catalytic intermediates, observations that provide new insight in the mechanism of OXO

Summary

Introduction

Oxalate oxidase is widespread in nature and has been found in bacteria [4], fungi [1, 5], and various plant tissues [6]. Glycolate Binding in the Crystal—The manganese ion at the active center of OXO is bound by three histidines, one glutamate, and two waters (Fig. 1b).

Results

Conclusion