Characterization of recombinant barley oxalate oxidase expressed by Pichia pastoris.
Oxalate oxidase catalyzes the oxidation of oxalate to carbon dioxide and hydrogen peroxide, making it useful for clinical analysis of oxalate in biological fluids. An artificial gene for barley oxalate oxidase has been used to produce functional recombinant enzyme in a Pichia pastoris heterologous expression system, yielding 250 mg of purified oxalate oxidase from 5 L of fermentation medium. The recombinant oxalate oxidase was expressed as a soluble, hexameric 140 kDa glycoprotein containing 0.2 g-atom Mn/monomer with a specific activity of 10 U/mg, similar to the properties reported for enzyme isolated from barley. No superoxide dismutase activity was detected in the recombinant oxalate oxidase. EPR spectra indicate that the majority of the manganese in the protein is present as Mn(II), and are consistent with the six-coordinate metal center reported in the recent X-ray crystal structure for barley oxalate oxidase. The EPR spectra change when bulky anions such as iodide bind, indicating conversion to a five-coordinate complex. Addition of oxalate perturbs the EPR spectrum of the Mn(II) sites, providing the first characterization of the substrate complex. The optical absorption spectrum of the concentrated protein contains features associated with a minor six-coordinate Mn(III) species, which disappears on addition of oxalate. EPR spin-trapping experiments indicate that carboxylate free radicals (CO2*-) are transiently produced by the enzyme in the presence of oxalate, most likely during reduction of the Mn(III) sites. These features are incorporated into a turnover mechanism for oxalate oxidase.
Whittaker
Mei M
MM
Department of Biochemistry and Molecular Biology, OGI School of Science and Engineering at Oregon Health & Science University, 20000 N.W. Walker Road, Beaverton, OR 97006-8921, USA.
Whittaker
James W
JW
D002264:Carboxylic Acids / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy / Q000379:methods; D004789:Enzyme Activation; D005285:Fermentation / Q000502:physiology; D005609:Free Radicals / Q000378:metabolism; D005813:Genes, Synthetic; D001467:Hordeum / Q000201:enzymology*; D008345:Manganese / Q000378:metabolism*; D010070:Oxalates / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D010843:Pichia / Q000235:genetics*; D011485:Protein Binding / Q000502:physiology; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013057:Spectrum Analysis / Q000379:methods; D013379:Substrate Specificity
D002264:Carboxylic Acids; D005609:Free Radicals; D010070:Oxalates; D011994:Recombinant Proteins; D008345:Manganese; D010088:Oxidoreductases; C020598:oxalate oxidase