title: Modulating O2 reactivity in a fungal flavoenzyme: involvement of aryl-alcohol oxidase Phe-501 contiguous to catalytic histidine.
abstract: Aryl-alcohol oxidase (AAO) is a flavoenzyme responsible for activation of O(2) to H(2)O(2) in fungal degradation of lignin. The AAO crystal structure shows a buried active site connected to the solvent by a hydrophobic funnel-shaped channel, with Phe-501 and two other aromatic residues forming a narrow bottleneck that prevents the direct access of alcohol substrates. However, ligand diffusion simulations show O(2) access to the active site following this channel. Site-directed mutagenesis of Phe-501 yielded a F501A variant with strongly reduced O(2) reactivity. However, a variant with increased reactivity, as shown by kinetic constants and steady-state oxidation degree, was obtained by substitution of Phe-501 with tryptophan. The high oxygen catalytic efficiency of F501W, ∼2-fold that of native AAO and ∼120-fold that of F501A, seems related to a higher O(2) availability because the turnover number was slightly decreased with respect to the native enzyme. Free diffusion simulations of O(2) inside the active-site cavity of AAO (and several in silico Phe-501 variants) yielded >60% O(2) population at 3-4 Å from flavin C4a in F501W compared with 44% in AAO and only 14% in F501A. Paradoxically, the O(2) reactivity of AAO decreased when the access channel was enlarged and increased when it was constricted by introducing a tryptophan residue. This is because the side chain of Phe-501, contiguous to the catalytic histidine (His-502 in AAO), helps to position O(2) at an adequate distance from flavin C4a (and His-502 Nε). Phe-501 substitution with a bulkier tryptophan residue resulted in an increase in the O(2) reactivity of this flavoenzyme.
authors.affiliation: Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain.
authors.forename: Aitor
authors.initials: A
authors.lastname: Hernández-Ortega
authors.forename: Fátima
authors.initials: F
authors.lastname: Lucas
authors.forename: Patricia
authors.initials: P
authors.lastname: Ferreira
authors.forename: Milagros
authors.initials: M
authors.lastname: Medina
authors.forename: Victor
authors.initials: V
authors.lastname: Guallar
authors.forename: Angel T
authors.initials: AT
authors.lastname: Martínez
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000465:Algorithms; D055162:Biocatalysis; D020134:Catalytic Domain; D019295:Computational Biology; D018360:Crystallography, X-Ray; D004058:Diffusion; D005415:Flavins / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D008024:Ligands; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D010100:Oxygen / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism*; D020076:Pleurotus / Q000201:enzymology*
chemical_list: D005415:Flavins; D008024:Ligands; D010649:Phenylalanine; D006639:Histidine; D000429:Alcohol Oxidoreductases; C077759:aryl-alcohol oxidase; D010100:Oxygen