Publications

• Oxalate oxidase from barley roots: purification to homogeneity and study of some molecular, catalytic, and binding properties.

  Kotsira V.P., Clonis Y.D.

  Oxalate oxidase (OXO) was purified to homogeneity in three steps from roots of barley seedlings. The purification method comprised: (i) thermal treatment (60 degrees C, 10 min), (ii) affinity chromatography on immobilized either Procion turquoise MX-G dye or biomimetic aminoethyl oxamic blue dye, ... >> More

  Oxalate oxidase (OXO) was purified to homogeneity in three steps from roots of barley seedlings. The purification method comprised: (i) thermal treatment (60 degrees C, 10 min), (ii) affinity chromatography on immobilized either Procion turquoise MX-G dye or biomimetic aminoethyl oxamic blue dye, and (iii) affinity chromatography on immobilized lectin concanavalin A (overall performance: 1096-fold purification, 42% recovery). The purified enzyme has a specific activity of 34 U mg-1 (25 degrees C), and is a homopentamer of M(r) approximately 125,000 (HPLC analysis) showing a single band on SDS-polyacryl-amide gel electrophoresis (M(r) approximately 26,000) after staining with silver nitrate. The kinetic constants of the purified enzyme for oxalate are K(m) 0.27 mM and kcat 22 s-1 (37 degrees C), whereas at [oxalate] > or = 4 mM the enzyme exhibited substrate inhibition. Barley root OXO contains no prosthetic group absorbing at 370 or 450 nm, and riboflavin and FAD have no effect on its activity. The enzyme is activated by 1 mM each of Ca2+ (1.7-fold) and Pb2+ (2.6-fold). Irreversible inactivation studies with denatured (70 degrees C) and native (37 degrees C) enzyme using the sulfhydryl-attacking reagent 5,5-dithiobis(2-nitrobenzoic) acid (1.4 mM), in the presence and absence of SDS, respectively, have shown that denatured OXO (4% SDS, 10 min, 100 degrees C) exhibited 10 HS groups per molecule, whereas native OXO displayed one accessible HS group per molecule after approximately 15 min incubation and, over the same period, maintained its catalytic activity to 90%. Furthermore, native OXO treated with beta-mercaptoethanol (1 mM) lost 83% of its catalytic activity within 5 min. These findings indicate that some cysteines may preserve the catalytic activity of OXO by maintaining the integrity of its tertiary structure via disulfide bond formation. << Less

  Arch Biochem Biophys 340:239-249(1997) [PubMed] [EuropePMC]

• Barley (Hordeum vulgare) oxalate oxidase is a manganese-containing enzyme.

  Requena L., Bornemann S.

  Oxalate oxidase (EC 1.2.3.4) catalyses the conversion of oxalate and dioxygen into CO(2) and H(2)O(2). The barley (Hordeum vulgare) seedling root enzyme was purified to homogeneity and shown by metal analysis and EPR spectroscopy to contain Mn(II) at up to 0.80 atom per subunit. The involvement of ... >> More

  Oxalate oxidase (EC 1.2.3.4) catalyses the conversion of oxalate and dioxygen into CO(2) and H(2)O(2). The barley (Hordeum vulgare) seedling root enzyme was purified to homogeneity and shown by metal analysis and EPR spectroscopy to contain Mn(II) at up to 0.80 atom per subunit. The involvement of Mn and neither flavin, Cu nor Fe in the direct conversion of dioxygen to H(2)O(2) makes oxalate oxidase unique. A model of the active site of the holoenzyme based on a homology model of the apoenzyme is proposed. << Less

  Biochem J 343:185-190(1999) [PubMed] [EuropePMC]