Substrate diffusion and oxidation in GMC oxidoreductases: an experimental and computational study on fungal aryl-alcohol oxidase
2011; Portland Press; Volume: 436; Issue: 2 Linguagem: Inglês
10.1042/bj20102090
ISSN1470-8728
AutoresAitor Hernández‐Ortega, Kenneth Borrelli, Patrícia Ferreira, Milagros Medina, Ángel T. Martı́nez, Vı́ctor Guallar,
Tópico(s)Polyamine Metabolism and Applications
ResumoAAO (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.
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