title: Characterization of three pyranose dehydrogenase isoforms from the litter-decomposing basidiomycete Leucoagaricus meleagris (syn. Agaricus meleagris).
abstract: Multigenicity is commonly found in fungal enzyme systems, with the purpose of functional compensation upon deficiency of one of its members or leading to enzyme isoforms with new functionalities through gene diversification. Three genes of the flavin-dependent glucose-methanol-choline (GMC) oxidoreductase pyranose dehydrogenase (AmPDH) were previously identified in the litter-degrading fungus Agaricus (Leucoagaricus) meleagris, of which only AmPDH1 was successfully expressed and characterized. The aim of this work was to study the biophysical and biochemical properties of AmPDH2 and AmPDH3 and compare them with those of AmPDH1. AmPDH1, AmPDH2 and AmPDH3 showed negligible oxygen reactivity and possess a covalently tethered FAD cofactor. All three isoforms can oxidise a range of different monosaccarides and oligosaccharides including glucose, mannose, galactose and xylose, which are the main constituent sugars of cellulose and hemicelluloses, and judging from the apparent steady-state kinetics determined for these sugars, the three isoforms do not show significant differences pertaining to their reaction with sugar substrates. They oxidize glucose both at C2 and C3 and upon prolonged reaction C2 and C3 double-oxidized glucose is obtained, confirming that the A. meleagris genes pdh2 (AY753308.1) and pdh3 (DQ117577.1) indeed encode CAZy class AA3_2 pyranose dehydrogenases. While reactivity with electron donor substrates was comparable for the three AmPDH isoforms, their kinetic properties differed significantly for the model electron acceptor substrates tested, a radical (the 2,2'-azino-bis[3-ethylbenzothiazoline-6-sulphonic acid] cation radical), a quinone (benzoquinone) and a complexed iron ion (the ferricenium ion). Thus, a possible explanation for this PDH multiplicity in A. meleagris could be that different isoforms react preferentially with structurally different electron acceptors in vivo.
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria.|BioToP-The Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Michael M H
authors.initials: MM
authors.lastname: Graf
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Sandra
authors.initials: S
authors.lastname: Weber
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria.|BioToP-The Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Kracher
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Roman
authors.initials: R
authors.lastname: Kittl
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Christoph
authors.initials: C
authors.lastname: Sygmund
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria.|BioToP-The Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Roland
authors.initials: R
authors.lastname: Ludwig
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria.|BioToP-The Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, 1190, Vienna, Austria.
authors.forename: Clemens
authors.initials: C
authors.lastname: Peterbauer
authors.affiliation: Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences (BOKU), Muthgasse 18, 1190, Vienna, Austria. dietmar.haltrich@boku.ac.at.|BioToP-The Doctoral Programme on Biomolecular Technology of Proteins, Muthgasse 18, 1190, Vienna, Austria. dietmar.haltrich@boku.ac.at.
authors.forename: Dietmar
authors.identifier: http://orcid.org/0000-0002-8722-8176
authors.initials: D
authors.lastname: Haltrich
mesh_terms: D000364:Agaricus / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D002482:Cellulose / Q000378:metabolism*; D005656:Fungal Proteins / Q000235:genetics; D005690:Galactose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D005810:Multigene Family / Q000235:genetics; D010084:Oxidation-Reduction; D013379:Substrate Specificity; D014994:Xylose / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D007527:Isoenzymes; D002482:Cellulose; D014994:Xylose; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase; D005947:Glucose; D005690:Galactose
keywords: 6-Hydroxy-FAD; Agaricus meleagris; Enzyme isoform; Enzyme multiplicity; Flavoprotein; Pyranose dehydrogenase