Modulating O2 reactivity in a fungal flavoenzyme: involvement of aryl-alcohol oxidase Phe-501 contiguous to catalytic histidine |
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Authors: | Hernández-Ortega Aitor Lucas Fátima Ferreira Patricia Medina Milagros Guallar Victor Martínez Angel T |
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Institution: | Centro de Investigaciones Biológicas, Consejo Superior de Investigaciones Científicas, Ramiro de Maeztu 9, E-28040 Madrid, Spain. |
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Abstract: | Aryl-alcohol oxidase (AAO) is a flavoenzyme responsible for activation of O2 to H2O2 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 O2 access to the active site following this channel. Site-directed mutagenesis of Phe-501 yielded a F501A variant with strongly reduced O2 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 O2 availability because the turnover number was slightly decreased with respect to the native enzyme. Free diffusion simulations of O2 inside the active-site cavity of AAO (and several in silico Phe-501 variants) yielded >60% O2 population at 3–4 ? from flavin C4a in F501W compared with 44% in AAO and only 14% in F501A. Paradoxically, the O2 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 O2 at an adequate distance from flavin C4a (and His-502 N?). Phe-501 substitution with a bulkier tryptophan residue resulted in an increase in the O2 reactivity of this flavoenzyme. |
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Keywords: | Computational Biology Crystal Structure Docking Enzyme Kinetics Enzyme Mechanisms Flavoproteins Lignin Degradation Oxygen Diffusion Site-directed Mutagenesis GMC Oxidoreductases |
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