Purification, characterization, and molecular cloning of a pyranose oxidase from the fruit body of the basidiomycete, Tricholoma matsutake.
A new H(2)O(2)-generating pyranose oxidase was purified as a strong antifungal protein from an arbuscular mycorrhizal fungus, Tricholoma matsutake. The protein showed a molecular mass of 250 kDa in gel filtration, and probably consisted of four identical 62 kDa subunits. The protein contained flavin moiety and it oxidized D-glucose at position C-2. H(2)O(2) and D-glucosone produced by the pyranose oxidase reaction showed antifungal activity, suggesting these compounds were the molecular basis of the antifungal property. The V(max), K(m), and k(cat) for D-glucose were calculated to be 26.6 U/mg protein, 1.28 mM, and 111/s, respectively. The enzyme was optimally active at pH 7.5 to 8.0 and at 50 degrees C. The preferred substrate was D-glucose, but 1,5-anhydro-D-glucitol, L-sorbose, and D-xylose were also oxidized at a moderate level. The cDNA encodes a protein consisting of 564 amino acids, showing 35.1% identity to Coriolus versicolor pyranose oxidase. The recombinant protein was used for raising the antibody.
Takakura
Yoshimitsu
Y
Plant Breeding and Genetics Research Laboratory, Japan Tobacco, Inc.. Iwata, Shizuoka, Japan. yoshimitsu.takakura@ims.jti.co.jp
Kuwata
Shigeru
S
D000595:Amino Acid Sequence; D000935:Antifungal Agents / Q000494:pharmacology; D001487:Basidiomycota / Q000201:enzymology / Q000235:genetics*; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D003001:Cloning, Molecular; D005658:Fungi / Q000187:drug effects*; D015723:Gene Library; D005947:Glucose / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D007661:Ketoses / Q000378:metabolism; D008969:Molecular Sequence Data; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics* / Q000494:pharmacology; D013013:Sorbose / Q000378:metabolism; D013379:Substrate Specificity; D014994:Xylose / Q000378:metabolism
D000935:Antifungal Agents; D007661:Ketoses; D021122:Protein Subunits; C017022:glucosone; D014994:Xylose; D006861:Hydrogen Peroxide; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D005947:Glucose; D013013:Sorbose