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Aromatic stacking interactions govern catalysis in aryl‐alcohol oxidase
Author(s) -
Ferreira Patricia,
HernándezOrtega Aitor,
Lucas Fátima,
Carro Juan,
Herguedas Beatriz,
Borrelli Kenneth W.,
Guallar Victor,
Martínez Angel T.,
Medina Milagros
Publication year - 2015
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.13221
Subject(s) - chemistry , stacking , aryl , stereochemistry , photochemistry , organic chemistry , alkyl
Aryl‐alcohol oxidase ( AAO , EC 1.1.3.7 ) generates H 2 O 2 for lignin degradation at the expense of benzylic and other π system‐containing primary alcohols, which are oxidized to the corresponding aldehydes. Ligand diffusion studies on Pleurotus eryngii AAO showed a T‐shaped stacking interaction between the Tyr92 side chain and the alcohol substrate at the catalytically competent position for concerted hydride and proton transfers. Bi‐substrate kinetics analysis revealed that reactions with 3‐chloro‐ or 3‐fluorobenzyl alcohols (halogen substituents) proceed via a ping–pong mechanism. However, mono‐ and dimethoxylated substituents (in 4‐methoxybenzyl and 3,4‐dimethoxybenzyl alcohols) altered the mechanism and a ternary complex was formed. Electron‐withdrawing substituents resulted in lower quantum mechanics stacking energies between aldehyde and the tyrosine side chain, contributing to product release, in agreement with the ping–pong mechanism observed in 3‐chloro‐ and 3‐fluorobenzyl alcohol kinetics analysis. In contrast, the higher stacking energies when electron donor substituents are present result in reaction of O 2 with the flavin through a ternary complex, in agreement with the kinetics of methoxylated alcohols. The contribution of Tyr92 to the AAO reaction mechanism was investigated by calculation of stacking interaction energies and site‐directed mutagenesis. Replacement of Tyr92 by phenylalanine does not alter the AAO kinetic constants (on 4‐methoxybenzyl alcohol), most probably because the stacking interaction is still possible. However, introduction of a tryptophan residue at this position strongly reduced the affinity for the substrate (i.e. the pre‐steady state K d and steady‐state K m increase by 150‐fold and 75‐fold, respectively), and therefore the steady‐state catalytic efficiency, suggesting that proper stacking is impossible with this bulky residue. The above results confirm the role of Tyr92 in substrate binding, thus governing the kinetic mechanism in AAO .