Publication: Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates
Issued Date
2015-11-01
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ISSN
17424658
1742464X
1742464X
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2-s2.0-84945457651
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Mahidol University
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SCOPUS
Bibliographic Citation
FEBS Journal. Vol.282, No.21 (2015), 4218-4241
Suggested Citation
Michael M.H. Graf, Jeerus Sucharitakul, Urban Bren, Dinh Binh Chu, Gunda Koellensperger, Stephan Hann, Paul G. Furtmüller, Christian Obinger, Clemens K. Peterbauer, Chris Oostenbrink, Pimchai Chaiyen, Dietmar Haltrich Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates. FEBS Journal. Vol.282, No.21 (2015), 4218-4241. doi:10.1111/febs.13417 Retrieved from: https://repository.li.mahidol.ac.th/handle/20.500.14594/35355
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Title
Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates
Abstract
© 2015 The Authors. FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. 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 105, 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.