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Reaction of pyranose dehydrogenase from Agaricus meleagris with its carbohydrate substrates.
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- المؤلفون: Graf, Michael M.H.1; Sucharitakul, Jeerus2; Bren, Urban3,4; Chu, Dinh Binh5,6; Koellensperger, Gunda7; Hann, Stephan5; Furtmüller, Paul G.8; Obinger, Christian8; Peterbauer, Clemens K.1; Oostenbrink, Chris3; Chaiyen, Pimchai9; Haltrich, Dietmar1
- المصدر:
FEBS Journal. Nov2015, Vol. 282 Issue 21, p4218-4241. 24p.
- الموضوع:
- معلومة اضافية
- نبذة مختصرة :
Monomeric Agaricus meleagris pyranose dehydrogenase ( Am PDH) belongs to the glucose-methanol-choline family of oxidoreductases. An FAD cofactor is covalently tethered to His103 of the enzyme. Am PDH 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 Am PDH 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 Am PDH 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 Am PDH. 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. [ABSTRACT FROM AUTHOR]
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