title: Analysis of Agaricus meleagris pyranose dehydrogenase N-glycosylation sites and performance of partially non-glycosylated enzymes.
abstract: Pyranose Dehydrogenase 1 from the basidiomycete Agaricus meleagris (AmPDH1) is an oxidoreductase capable of oxidizing a broad variety of sugars. Due to this and its ability of dioxidation of substrates and no side production of hydrogen peroxide, it is studied for use in enzymatic bio-fuel cells. In-vitro deglycosylated AmPDH1 as well as knock-out mutants of the N-glycosylation sites N75and N175, near the active site entrance, were previously shown to improve achievable current densities of graphite electrodes modified with AmPDH1 and an osmium redox polymer acting as a redox mediator, up to 10-fold. For a better understanding of the role of N-glycosylation of AmPDH1, a systematic set of N-glycosylation site mutants was investigated in this work, regarding expression efficiency, enzyme activity and stability. Furthermore, the site specific extend of N-glycosylation was compared between native and recombinant wild type AmPDH1. Knocking out the site N252prevented the attachment of significantly extended N-glycan structures as detected on polyacrylamide gel electrophoresis, but did not significantly alter enzyme performance on modified electrodes. This suggests that not the molecule size but other factors like accessibility of the active site improved performance of deglycosylated AmPDH1/osmium redox polymer modified electrodes. A fourth N-glycosylation site of AmPDH1 could be confirmed by mass spectrometry at N319, which appeared to be conserved in related fungal pyranose dehydrogenases but not in other members of the glucose-methanol-choline oxidoreductase structural family. This site was shown to be the only one that is essential for functional recombinant expression of the enzyme.
authors.affiliation: Department of Food Sciences and Technology, BOKU-University of Natural Resources and Life Sciences Vienna, Muthgasse 18, A-1190 Wien, Austria; Department of Analytical Chemistry/Biochemistry and Structural Biology, Lund University, PO Box 124, SE-221 00 Lund, Sweden.
authors.forename: Christoph
authors.initials: C
authors.lastname: Gonaus
authors.affiliation: Department of Chemistry, BOKU-University of Natural Resources and Life Sciences Vienna, Muthgasse 18, A-1190 Wien, Austria.
authors.forename: Daniel
authors.initials: D
authors.lastname: Maresch
authors.affiliation: Department of Food Sciences and Technology, BOKU-University of Natural Resources and Life Sciences Vienna, Muthgasse 18, A-1190 Wien, Austria; Department of Analytical Chemistry/Biochemistry and Structural Biology, Lund University, PO Box 124, SE-221 00 Lund, Sweden.
authors.forename: Katharina
authors.initials: K
authors.lastname: Schropp
authors.affiliation: School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland.
authors.forename: Peter
authors.initials: P
authors.lastname: Ó Conghaile
authors.affiliation: School of Chemistry, National University of Ireland Galway, University Road, Galway, Ireland.
authors.forename: Dónal
authors.initials: D
authors.lastname: Leech
authors.affiliation: Department of Analytical Chemistry/Biochemistry and Structural Biology, Lund University, PO Box 124, SE-221 00 Lund, Sweden.
authors.forename: Lo
authors.initials: L
authors.lastname: Gorton
authors.affiliation: Department of Food Sciences and Technology, BOKU-University of Natural Resources and Life Sciences Vienna, Muthgasse 18, A-1190 Wien, Austria. Electronic address: clemens.peterbauer@boku.ac.at.
authors.forename: Clemens K
authors.initials: CK
authors.lastname: Peterbauer
mesh_terms: D000364:Agaricus / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D001665:Binding Sites / Q000235:genetics; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D004795:Enzyme Stability; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism
chemical_list: D005656:Fungal Proteins; D011994:Recombinant Proteins; D002237:Carbohydrate Dehydrogenases; C022489:pyranose oxidase
keywords: Agaricus meleagris; Enzymatic biofuel cell; N-glycosylation; Pyranose dehydrogenase