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Please use this identifier to cite or link to this item: http://hdl.handle.net/10261/49233
Title: Substrate diffusion and oxidation in GMC oxidoreductases: an experimental and computational study on fungal aryl-alcohol oxidase
Authors: Hernández-Ortega, Aitor; Borrelli, Kenneth; Ferreira, Patricia; Medina, Milagros; Martínez, Ángel T.; Guallar, Victor
Keywords: aryl-alcohol oxidase (AAO)
catalytic base
glucose-methanol-choline oxidase (GMC) oxidoreductase
molecular docking
quantum mechanics/molecular mechanics (QM/MM)
reaction mechanism
Issue Date: 4-Mar-2011
Publisher: Portland Press
Citation: Biochemical Journal 436: 341-350(2011)
Abstract: AAO (aryl-alcohol oxidase) provides H2O2 in fungal degradation of lignin, a process of high biotechnological interest. The crystal structure of AAO does not show open access to the active site, where different aromatic alcohols are oxidized. In the present study we investigated substrate diffusion and oxidation in AAO compared with the structurally related CHO (choline oxidase). Cavity finder and ligand diffusion simulations indicate the substrate-entrance channel, requiring side-chain displacements and involving a stacking interaction with Tyr92. Mixed QM (quantum mechanics)/MM (molecular mechanics) studies combined with site-directed mutagenesis showed two active-site catalytic histidine residues, whose substitution strongly decreased both catalytic and transient-state reduction constants for p-anisyl alcohol in the H502A (over 1800-fold) and H546A (over 35-fold) variants. Combination of QM/MM energy profiles, protonation predictors, molecular dynamics, mutagenesis and pH profiles provide a robust answer regarding the nature of the catalytic base. The histidine residue in front of the FAD ring, AAO His502 (and CHO His466), acts as a base. For the two substrates assayed, it was shown that proton transfer preceded hydride transfer, although both processes are highly coupled. No stable intermediate was observed in the energy profiles, in contrast with that observed for CHO. QM/MM, together with solvent KIE (kinetic isotope effect) results, suggest a non-synchronous concerted mechanism for alcohol oxidation by AAO
Description: 10 páginas, 7 figuras, 2 tablas -- PAGS nros. 341-350
Publisher version (URL): http://dx.doi.org/10.1042/BJ20102090
URI: http://hdl.handle.net/10261/49233
DOI: 10.1042/BJ20102090
ISSN: 0264-6021
E-ISSN: 1470-8728
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