Document Detail

Substrate diffusion and oxidation in GMC oxidoreductases: An experimental and computational study on fungal aryl-alcohol oxidase.
MedLine Citation:
PMID:  21375505     Owner:  NLM     Status:  Publisher    
Aryl-alcohol oxidase (AAO) provides H2O2 in fungal degradation of lignin, a process of high biotechnological interest. Its crystal structure does not show an open access to the active site, where different aromatic alcohols are oxidized. We studied substrate diffusion and oxidation in AAO, compared with the structurally-related choline oxidase (CHO). Cavity finder and ligand diffusion simulations indicate the substrate entrance channel, requiring side-chain displacements and involving a stacking interaction with Tyr92. Mixed quantum mechanics/molecular mechanics (QM/MM) studies combined with site-directed mutagenesis showed two active-site catalytic histidines, 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 give a robust answer about the nature of the catalytic base. The histidine 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 results, suggest a non-synchronous concerted mechanism for alcohol oxidation by AAO.
Aitor Hernández-Ortega; Kenneth Borrelli; Patricia Ferreira; Milagros Medina; Angel T Martinez; Victor Guallar
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Publication Detail:
Type:  JOURNAL ARTICLE     Date:  2011-3-4
Journal Detail:
Title:  The Biochemical journal     Volume:  -     ISSN:  1470-8728     ISO Abbreviation:  -     Publication Date:  2011 Mar 
Date Detail:
Created Date:  2011-3-7     Completed Date:  -     Revised Date:  -    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  -    
Other Details:
Languages:  ENG     Pagination:  -     Citation Subset:  -    
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