Modulating O2 reactivity in a fungal flavoenzyme: involvement of aryl-alcohol oxidase Phe-501 contiguous to catalytic histidine
| Autor: | Aitor, Hernández-Ortega, Fátima, Lucas, Patricia, Ferreira, Milagros, Medina, Victor, Guallar, Angel T, Martínez |
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| Rok vydání: | 2011 |
| Předmět: |
Models
Molecular Phenylalanine fungi technology industry and agriculture food and beverages Computational Biology macromolecular substances Crystallography X-Ray Ligands Pleurotus complex mixtures Diffusion Oxygen Alcohol Oxidoreductases Kinetics Catalytic Domain Flavins Mutation Biocatalysis Mutagenesis Site-Directed Enzymology Histidine Oxidation-Reduction Algorithms |
| Zdroj: | The Journal of biological chemistry. 286(47) |
| ISSN: | 1083-351X |
| Popis: | 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) yielded60% 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. |
| Databáze: | OpenAIRE |
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