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Título : Modulating O2 reactivity in a fungal flavoenzyme: Involvement of aryl-alcohol oxidase Phe-501 contiguous to catalytic histidine
Autor : Hernández-Ortega, Aitor, Lucas, Fátima, Ferreira, Patricia, Medina, Milagros, Guallar, Victor, Martínez Ferrer, Ángel Tomás
Palabras clave : Computational Biology
Crystal Structure
Enzyme Kinetics
Enzyme Mechanisms
Lignin Degradation
Oxygen Diffusion
Site-directed Mutagenesis
GMC Oxidoreductases
Fecha de publicación : 25-Nov-2011
Editor: American Society for Biochemistry and Molecular Biology
Resumen: 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 Å 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 Nϵ). Phe-501 substitution with a bulkier tryptophan residue resulted in an increase in the O2 reactivity of this flavoenzyme
Descripción : 10 páginas, 6 figuras, 2 tablas -- PAGS nros. 41105-41114
Versión del editor:
ISSN: 0021-9258
DOI: 10.1074/jbc.M111.282467
Fecha de embargo: 2012-11-25
Citación : Journal of Biological Chemistry 286: 41105-41114(2011)
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