title: Agaricus meleagris pyranose dehydrogenase: influence of covalent FAD linkage on catalysis and stability.
abstract: Pyranose dehydrogenase (PDH) is a monomeric flavoprotein belonging to the glucose-methanol-choline (GMC) family of oxidoreductases. It catalyzes the oxidation of free, non-phosphorylated sugars to the corresponding keto sugars. The enzyme harbors an FAD cofactor that is covalently attached to histidine 103 via an 8α-N(3) histidyl linkage. Our previous work showed that variant H103Y was still able to bind FAD (non-covalently) and perform catalysis but steady-state kinetic parameters for several substrates were negatively affected. In order to investigate the impact of the covalent FAD attachment in Agaricus meleagris PDH in more detail, pre-steady-state kinetics, reduction potential and stability of the variant H103Y in comparison to the wild-type enzyme were probed. Stopped-flow analysis revealed that the mutation slowed down the reductive half-reaction by around three orders of magnitude whereas the oxidative half-reaction was affected only to a minor degree. This was reflected by a decrease in the standard reduction potential of variant H103Y compared to the wild-type protein. The existence of an anionic semiquinone radical in the resting state of both the wild-type and variant H103Y was demonstrated using electron paramagnetic resonance (EPR) spectroscopy and suggested a higher mobility of the cofactor in the variant H103Y. Unfolding studies showed significant negative effects of the disruption of the covalent bond on thermal and conformational stability. The results are discussed with respect to the role of covalently bound FAD in catalysis and stability.
authors.affiliation: Department of Food Science and Technology, Food Biotechnology Laboratory, BOKU - University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Iris
authors.initials: I
authors.lastname: Krondorfer
authors.affiliation: Department of Food Science and Technology, Food Biotechnology Laboratory, BOKU - University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Dagmar
authors.initials: D
authors.lastname: Brugger
authors.affiliation: Department of Food Science and Technology, Food Biotechnology Laboratory, BOKU - University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Regina
authors.initials: R
authors.lastname: Paukner
authors.affiliation: Department of Food Science and Technology, Food Biotechnology Laboratory, BOKU - University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Scheiblbrandner
authors.affiliation: Department of Chemistry, Division of Biochemistry, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Katharina F
authors.initials: KF
authors.lastname: Pirker
authors.affiliation: Department of Chemistry, Division of Biochemistry, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hofbauer
authors.affiliation: Department of Chemistry, Division of Biochemistry, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Paul G
authors.initials: PG
authors.lastname: Furtmüller
authors.affiliation: Department of Chemistry, Division of Biochemistry, BOKU - University of Natural Resources and Life Sciences, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Christian
authors.initials: C
authors.lastname: Obinger
authors.affiliation: Department of Food Science and Technology, Food Biotechnology Laboratory, BOKU - University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Dietmar
authors.initials: D
authors.lastname: Haltrich
authors.affiliation: Department of Food Science and Technology, Food Biotechnology Laboratory, BOKU - University of Natural Resources and Life Sciences, Muthgasse 11, 1190 Vienna, Austria. Electronic address: clemens.peterbauer@boku.ac.at.
authors.forename: Clemens K
authors.initials: CK
authors.lastname: Peterbauer
mesh_terms: D000364:Agaricus / Q000201:enzymology*; D016227:Benzoquinones / Q000378:metabolism; D055162:Biocatalysis; D050260:Carbohydrate Metabolism; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism*; D010084:Oxidation-Reduction; D010088:Oxidoreductases / Q000737:chemistry* / Q000378:metabolism*; D011487:Protein Conformation; D013696:Temperature
chemical_list: D016227:Benzoquinones; D005182:Flavin-Adenine Dinucleotide; C025232:semiquinone radicals; D010088:Oxidoreductases
keywords: Conformational stability; Covalent flavinylation; FAD; Pyranose dehydrogenase; Semiquinone radical; Thermal stability