Screening of basidiomycete fungi for the quinone-dependent sugar C-2/C-3 oxidoreductase, pyranose dehydrogenase, and properties of the enzyme from Macrolepiota rhacodes.
Mycelial cultures of 76 strains of lignocellulose-degrading basidiomycete fungi were screened for the activity of pyranose dehydrogenase, a novel sugar oxidoreductase recently detected in Agaricus bisporus. Of these fungi, 37 strains belonging to seven phylogenetically related genera of mostly litter-decomposing Agaricales were positive for the dehydrogenase, based on activity assays towards D-glucose with 1,4-benzoquinone or ferricenium ion as electron acceptors, and on TLC/HPLC analyses of the reaction products. Lack of activity with O(2) as the oxidant, specificity for C-3 of D-glucose, and active extracellular secretion of the enzyme were used as criteria to differentiate pyranose dehydrogenase from pyranose 2-oxidase (EC 1.1.3.10), known to be produced by numerous wood-rotting fungi. Extracellular pyranose dehydrogenase from Macrolepiota rhacodes was heavily glycosylated. The enzyme was characterized as a 78-kDa flavoprotein under denaturing conditions and a 76-kDa native protein using gel filtration. This enzyme had a maximum extracellular activity of 4.1 U ml(-1) in 39-day liquid cultures. It exhibited broad selectivity for sugar substrates and oxidized D-glucose (K(m)=1.82) exclusively at C-3 to 3-dehydro-D-glucose (D-ribo-hexos-3-ulose), in contrast to pyranose dehydrogenases from Agaricus species, which acted at both C-3 and C-2 of D-glucose. The N-terminal sequence, AVVYRHPDEL, showed significant similarity with that reported for A. bisporus.
Volc
J
J
Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídenská 1083, 142 20 Prague 4, Czech Republic. volc@biomed.cas.cz
Kubátová
E
E
Daniel
G
G
Sedmera
P
P
Haltrich
D
D
D001487:Basidiomycota / Q000201:enzymology* / Q000254:growth & development; D016227:Benzoquinones / Q000378:metabolism; D002237:Carbohydrate Dehydrogenases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D050260:Carbohydrate Metabolism; D002482:Cellulose / Q000378:metabolism; D005420:Flavoproteins / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008031:Lignin / Q000378:metabolism; D008970:Molecular Weight; D010088:Oxidoreductases / Q000032:analysis / Q000737:chemistry / Q000378:metabolism*; D018517:Plant Roots / Q000382:microbiology
D016227:Benzoquinones; D005420:Flavoproteins; C036909:lignocellulose; C004532:quinone; D002482:Cellulose; D008031:Lignin; D010088:Oxidoreductases; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D005947:Glucose