Inversion of stereospecificity of vanillyl-alcohol oxidase
AUTOR(ES)
van den Heuvel, Robert H. H.
FONTE
The National Academy of Sciences
RESUMO
Vanillyl-alcohol oxidase (VAO) is the prototype of a newly recognized family of structurally related oxidoreductases sharing a conserved FAD-binding domain. The active site of VAO is formed by a cavity where the enzyme is able to catalyze many reactions with phenolic substrates. Among these reactions is the stereospecific hydroxylation of 4-ethylphenol-forming (R)-1-(4′-hydroxyphenyl)ethanol. During this conversion, Asp-170 is probably critical for the hydration of the initially formed p-quinone methide intermediate. By site-directed mutagenesis, the putative active site base has been relocated to the opposite face of the active site cavity. In this way, a change in stereospecificity has been achieved. Like native VAO, the single mutants T457E, D170A, and D170S preferentially converted 4-ethylphenol to the (R)-enantiomer of 1-(4′-hydroxyphenyl)ethanol. The double mutants D170A/T457E and D170S/T457E exhibited an inverted stereospecificity with 4-ethylphenol. Particularly, D170S/T457E was strongly (S)-selective, with an enantiomeric excess of 80%. The crystal structure of D170S/T457E, in complex with trifluoromethylphenol, showed a highly conserved mode of ligand binding and revealed that the distinctive catalytic properties of this mutant are not caused by major structural changes.
ACESSO AO ARTIGO
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=16885Documentos Relacionados
- Regio- and Stereospecific Conversion of 4-Alkylphenols by the Covalent Flavoprotein Vanillyl-Alcohol Oxidase
- Structural determinants of stereospecificity in yeast alcohol dehydrogenase.
- Xylosylation of Phenolic Hydroxyl Groups of the Monomeric Lignin Model Compounds 4-Methylguaiacol and Vanillyl Alcohol by Coriolus versicolor
- Substrate specificity and stereospecificity of nicotinamide adenine dinucleotide-linked alcohol dehydrogenases from methanol-grown yeasts.
- Production of Alcohol Oxidase by Several Basidiomycetes