Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates.
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.
Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences Vienna (BOKU), Austria.
Michael M H
MM
Graf
Department of Biochemistry, Faculty of Dentistry, Chulalongkorn University, Bangkok, Thailand.
Jeerus
J
Sucharitakul
Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences Vienna (BOKU), Austria.|Laboratory for Physical Chemistry and Chemical Thermodynamics, Faculty of Chemistry and Chemical Technology, University of Maribor, Slovenia.
Urban
U
Bren
Division of Analytical Chemistry, Department of Chemistry, University of Natural Resources and Life Sciences Vienna (BOKU), Austria.|School of Chemical Engineering, Department of Analytical Chemistry, Hanoi University of Science and Technology, Hanoi, Vietnam.
Dinh Binh
DB
Chu
Institute of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Austria.
Gunda
G
Koellensperger
Division of Analytical Chemistry, Department of Chemistry, University of Natural Resources and Life Sciences Vienna (BOKU), Austria.
Stephan
S
Hann
Division of Biochemistry, Department of Chemistry, University of Natural Resources and Life Sciences Vienna (BOKU), Austria.
Paul G
PG
Furtmüller
Division of Biochemistry, Department of Chemistry, University of Natural Resources and Life Sciences Vienna (BOKU), Austria.
Christian
C
Obinger
Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences Vienna (BOKU), Austria.
Clemens K
CK
Peterbauer
Institute of Molecular Modeling and Simulation, University of Natural Resources and Life Sciences Vienna (BOKU), Austria.
Chris
C
Oostenbrink
Department of Biochemistry and Center of Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, Bangkok, Thailand.
Pimchai
P
Chaiyen
Food Biotechnology Laboratory, Department of Food Science and Technology, University of Natural Resources and Life Sciences Vienna (BOKU), Austria.
Dietmar
D
Haltrich
D000364:Agaricus / Q000201:enzymology* / Q000235:genetics; D019943:Amino Acid Substitution; D002237:Carbohydrate Dehydrogenases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D020134:Catalytic Domain / Q000235:genetics; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005182:Flavin-Adenine Dinucleotide / Q000737:chemistry / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008401:Gas Chromatography-Mass Spectrometry; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D056004:Molecular Dynamics Simulation; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity
D005656:Fungal Proteins; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; D002237:Carbohydrate Dehydrogenases; D005947:Glucose
Agaricus meleagris pyranose dehydrogenase; enzyme kinetics; flavin-dependent oxidoreductase; glucose-methanol-choline family of oxidoreductases; molecular dynamics simulations