title: Aromatic stacking interactions govern catalysis in aryl-alcohol oxidase.
abstract: Aryl-alcohol oxidase (AAO, EC 1.1.3.7) generates H2 O2 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 O2 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 Kd and steady-state Km 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.
authors: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, and Instituto de Biocomputación y Física de Sistemas Complejos, Zaragoza, Spain.
authors: Patricia
authors: P
authors: Ferreira
authors: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors: Aitor
authors: A
authors: Hernández-Ortega
authors: Joint Barcelona Supercomputing Center-Centre for Genomic Regulation-Institute for Research in Biomedicine Research Program in Computational Biology, Barcelona Supercomputing Center, Barcelona, Spain.
authors: Fátima
authors: F
authors: Lucas
authors: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors: Juan
authors: J
authors: Carro
authors: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, and Instituto de Biocomputación y Física de Sistemas Complejos, Zaragoza, Spain.
authors: Beatriz
authors: B
authors: Herguedas
authors: Joint Barcelona Supercomputing Center-Centre for Genomic Regulation-Institute for Research in Biomedicine Research Program in Computational Biology, Barcelona Supercomputing Center, Barcelona, Spain.
authors: Kenneth W
authors: KW
authors: Borrelli
authors: Joint Barcelona Supercomputing Center-Centre for Genomic Regulation-Institute for Research in Biomedicine Research Program in Computational Biology, Barcelona Supercomputing Center, Barcelona, Spain.|Institució Catalana de Recerca i Estudis Avançats, Barcelona, Spain.
authors: Victor
authors: V
authors: Guallar
authors: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Madrid, Spain.
authors: Angel T
authors: AT
authors: Martínez
authors: Departamento de Bioquímica y Biología Molecular y Celular, Facultad de Ciencias, Universidad de Zaragoza, and Instituto de Biocomputación y Física de Sistemas Complejos, Zaragoza, Spain.
authors: Milagros
authors: M
authors: Medina
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001483:Base Sequence; D001592:Benzyl Alcohols / Q000378:metabolism; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics; D004271:DNA, Fungal / Q000235:genetics; D005656:Fungal Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D020076:Pleurotus / Q000201:enzymology / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055672:Static Electricity; D013379:Substrate Specificity; D013816:Thermodynamics; D014443:Tyrosine / Q000737:chemistry
chemical_list: D001592:Benzyl Alcohols; D004271:DNA, Fungal; D005656:Fungal Proteins; D011994:Recombinant Proteins; D014443:Tyrosine; D000429:Alcohol Oxidoreductases; C077759:aryl-alcohol oxidase
keywords: GMC oxidoreductases; aromatic stacking; aryl-alcohol oxidase; catalytic mechanism; steady-state and pre-steady state kinetics