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

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.Oxalate oxidase (OXO; EC 1.2.3.4) 6 catalyzes the oxidation of oxalate, reducing dioxygen to hydrogen peroxide and forming 2 mol of carbonOxalate oxidase is widespread in nature and has been found in bacteria (4), fungi (1, 5), and various plant tissues (6). It has been detected in barley seedling roots during germination and in the leaves of mature barley plants in response to powdery mildew infection (6, 7), suggesting a role in plant signaling and defense. The enzyme has been purified to homogeneity from barley seedling roots and its N-terminal sequence determined, allowing the corresponding cDNA to be isolated and the complete primary sequence to be determined (3,8). These developments led to the recognition that the enzyme, OXO, is identical to an important marker of grain development during germination of wheat called germin (3,8). OXO is a member of a functionally diverse protein superfamily known as the cupins (9) or double stranded ␤-helix proteins (10). Barley OXO forms a hexamer that has extreme stability to heat and proteolysis (11).Spectroscopic studies demonstrated that OXO requires manganese for catalysis (12) and subsequent crystallographic studies on the barley enzyme revealed the structure of the hexamer (Fig. 1a) and confirmed the presence of a mononuclear manganese center buried deep within its jellyroll ␤-barrel domain (13). The manganese is bound by the side chains of three histidines and one glutamate residue, as well as two water molecules that occupy adjacent positions in the roughly octahedral metal complex (Fig. 1b). Based on the lack of obvious optical absorption and the presence of a characteristic EPR spectrum, the manganese ion has been assigned as the reduced Mn(II) oxidation state in the resting enzyme (12). Spectroscopic studies using recombinant OXO expressed in Pichia pastoris confirmed the presence of Mn(II) in the resting recombinant enzyme and provided the first spectroscopic evidence for oxalate binding to the manganese (14). The EPR signal of the anaerobic substrate complex, like that of the nativ...

Burst Kinetics and Redox Transformations of the Active Site Manganese Ion in Oxalate Oxidase

“…In the first step (Scheme 4, step 1), the active, resting Mn(III) enzyme binds substrate (as the monoanion) to form a Michaelis complex. Substrate is shown with monodentate carboxylate coordination, consistent with recent x-ray structural studies on a substrate analog (glycolate) complex, which also identifies a role for Asn 75 and Asn 85 in hydrogen bond stabilization of the complex (16). Under anaerobic conditions, oxalate has been shown to reduce the Mn(III) form of the enzyme (10) (Scheme 4, step 2).…”

“…Recent advances in x-ray structures of oxalate oxidase (9,15,16) and the availability of recombinant enzyme provides a foundation for detailed mechanistic studies on this interesting enzyme. In the present work, we have observed unusual nonstoichiometric burst kinetics, which has led to a clearer understanding of the role of the manganese ion in the catalytic reaction.…”

“…Structural characterization of the glycolate (substrate analog) complex of OXO suggest that two asparagine residues (Asn 75 and Asn 85 ) may play a role in orienting and stabilizing complexes of substrate or intermediates. Site-directed mutagenesis of these two residues in recombinant barley OXO demonstrates that they are essential for activity (16).…”

Oxalate Biominerals

“…The involvement of Mn(II) and no flavin, iron or copper in the direct conversion of O 2 to H 2 O 2 makes oxalate oxidase unique [216]. Some mechanistic studies have also been recently performed [215,218] and allow first insights into the reaction pathways.…”

Structure of cis-dichlorobis(1,10-phenanthroline)manganese(II) and cis-dichlorobis(2,2´-bipyridine)manganese(II)

“…Manganese complexes are of current interest in studies of molecular magnetism and in applications as magnetic recording (Ako et al, 2006;Aromi & Brechin, 2006) and manganese-enhanced MRI (Koretsky& Silva, 2004), and they can serve as model compounds of the bioinorganic chemistry of manganese (Deeth, 2008;Dismukes, 2006) including O 2 -evolving center of photosystem II (Umena et al, 2011) and various manganese-containing redox enzymes as dioxygenases (Georgiev et al, 2006), oxalate oxidase (Opaleye et al, 2006), catalase (Whittaker, 2012), superoxid dismutase (Friedel et al, 2004) and peroxidase (Sundaramoorthy et al, 2010). More manganese complexes especially for catalase and/or superoxide dismutase mimetics contain chelating N,N-donor ligands as 1,10-phenanthroline or 2,2´-bipyridine (Devereux et al, 1996;Geraghty et al, 1998;Kani et al, 2008;McCann et al, 1998;Viossat et al, 2003).…”