1. Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates.
- Author
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Graf MM, Sucharitakul J, Bren U, Chu DB, Koellensperger G, Hann S, Furtmüller PG, Obinger C, Peterbauer CK, Oostenbrink C, Chaiyen P, and Haltrich D
- Subjects
- Agaricus genetics, Amino Acid Substitution, Carbohydrate Dehydrogenases chemistry, Carbohydrate Dehydrogenases genetics, Catalytic Domain genetics, Crystallography, X-Ray, Enzyme Stability, Flavin-Adenine Dinucleotide chemistry, Flavin-Adenine Dinucleotide metabolism, Fungal Proteins chemistry, Fungal Proteins genetics, Gas Chromatography-Mass Spectrometry, Glucose metabolism, Kinetics, Models, Molecular, Molecular Dynamics Simulation, Mutagenesis, Site-Directed, Oxidation-Reduction, Recombinant Proteins chemistry, Recombinant Proteins genetics, Recombinant Proteins metabolism, Substrate Specificity, Agaricus enzymology, Carbohydrate Dehydrogenases metabolism, Fungal Proteins metabolism
- Abstract
Monomeric Agaricus meleagris pyranose dehydrogenase (AmPDH) belongs to the glucose-methanol-choline family of oxidoreductases. An FAD cofactor is covalently tethered to His103 of the enzyme. AmPDH can double oxidize various mono- and oligosaccharides at different positions (C1 to C4). To study the structure/function relationship of selected active-site residues of AmPDH pertaining to substrate (carbohydrate) turnover in more detail, several active-site variants were generated, heterologously expressed in Pichia pastoris, and characterized by biochemical, biophysical and computational means. The crystal structure of AmPDH shows two active-site histidines, both of which could take on the role as the catalytic base in the reductive half-reaction. Steady-state kinetics revealed that His512 is the only catalytic base because H512A showed a reduction in (kcat /KM )glucose by a factor of 10(5) , whereas this catalytic efficiency was reduced by two or three orders of magnitude for His556 variants (H556A, H556N). This was further corroborated by transient-state kinetics, where a comparable decrease in the reductive rate constant was observed for H556A, whereas the rate constant for the oxidative half-reaction (using benzoquinone as substrate) was increased for H556A compared to recombinant wild-type AmPDH. Steady-state kinetics furthermore indicated that Gln392, Tyr510, Val511 and His556 are important for the catalytic efficiency of PDH. Molecular dynamics (MD) simulations and free energy calculations were used to predict d-glucose oxidation sites, which were validated by GC-MS measurements. These simulations also suggest that van der Waals interactions are the main driving force for substrate recognition and binding., (© 2015 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)
- Published
- 2015
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