Pyranose 2-dehydrogenase, a novel sugar oxidoreductase from the basidiomycete fungus Agaricus bisporus.
A novel C-2-specific sugar oxidoreductase, tentatively designated as pyranose 2-dehydrogenase, was purified 68-fold to apparent homogeneity (16.4 U/mg protein) from the mycelia of Agaricus bisporus, which expressed maximum activity of the enzyme during idiophasic growth in liquid media. Using 1,4-benzoquinone as an electron acceptor, pyranose 2-dehydrogenase oxidized d-glucose to d-arabino-2-hexosulose (2-dehydroglucose, 2-ketoglucose), which was identified spectroscopically through its N,N-diphenylhydrazone. The enzyme is highly nonspecific. d-,l-Arabinose, d-ribose, d-xylose, d-galactose, and several oligosaccharides and glycopyranosides were all converted to the corresponding 2-aldoketoses (aldosuloses) as indicated by TLC. d-Glucono-1,5-lactone, d-arabino-2-hexosulose, and l-sorbose were also oxidized at significant rates. UV/VIS spectrum of the native enzyme (lambdamax 274, 362, and 465 nm) was consistent with a flavin prosthetic group. In contrast to oligomeric intracellular pyranose 2-oxidase (EC 1.1.3.10), pyranose 2-dehydrogenase is a monomeric glycoprotein (pI 4.2) incapable of reducing O2 to H2O2 (> 5 x 10(4)-fold lower rate using a standard pyranose oxidase assay); pyranose 2-dehydrogenase is actively secreted into the extracellular fluid (up to 0.5 U/ml culture filtrate). The dehydrogenase has a native molecular mass of approximately 79 kDa as determined by gel filtration; its subunit molecular mass is approximately 75 kDa as estimated by SDS-PAGE. Two pH optima of the enzyme were found, one alkaline at pH 9 (phosphate buffer) and the other acidic at pH 4 (acetate buffer). Ag+, Hg2+, Cu2+, and CN- (10 mM) were inhibitory, while 50 mM acetate had an activating effect.
Volc
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Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeònská 1083, 14220 Prague 4, Czech Republic.
Kubátová
E
E
Wood
D A
DA
Daniel
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G
D000364:Agaricus / Q000201:enzymology*; D001089:Arabinose / Q000378:metabolism; D016227:Benzoquinones / Q000494:pharmacology; D002237:Carbohydrate Dehydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism*; D003300:Copper / Q000494:pharmacology; D003486:Cyanides / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors; D005690:Galactose / Q000378:metabolism; D005942:Gluconates / Q000378:metabolism; D005947:Glucose / Q000031:analogs & derivatives / Q000378:metabolism; D006027:Glycosides / Q000378:metabolism; D006861:Hydrogen Peroxide / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007526:Isoelectric Point; D007661:Ketoses; D007783:Lactones; D008628:Mercury / Q000494:pharmacology; D009844:Oligosaccharides / Q000378:metabolism; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism; D012266:Ribose / Q000378:metabolism; D012834:Silver / Q000494:pharmacology; D013013:Sorbose / Q000378:metabolism; D014994:Xylose / Q000378:metabolism
D016227:Benzoquinones; D003486:Cyanides; D004791:Enzyme Inhibitors; D005942:Gluconates; D006027:Glycosides; D007661:Ketoses; D007783:Lactones; D009844:Oligosaccharides; C017022:glucosone; D012834:Silver; C004532:quinone; D012266:Ribose; D003300:Copper; D014994:Xylose; D001089:Arabinose; D006861:Hydrogen Peroxide; D002237:Carbohydrate Dehydrogenases; D008628:Mercury; D005947:Glucose; D013013:Sorbose; D010100:Oxygen; C010730:beta-glucono-1,5-lactone; D005690:Galactose