Inversion of stereospecificity of vanillyl-alcohol oxidase.
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.
Department of Biomolecular Sciences, Laboratory of Biochemistry, Wageningen University, The Netherlands.
R H
RH
van Den Heuvel
M W
MW
Fraaije
M
M
Ferrer
A
A
Mattevi
W J
WJ
van Berkel
D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution / Q000235:genetics; D001224:Aspartic Acid / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D018698:Glutamic Acid / Q000235:genetics / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D006900:Hydroxylation; D007700:Kinetics; D008958:Models, Molecular; D009154:Mutation / Q000235:genetics; D010407:Penicillium / Q000201:enzymology*; D010636:Phenols / Q000378:metabolism; D010626:Phenylethyl Alcohol / Q000031:analogs & derivatives* / Q000378:metabolism; D011487:Protein Conformation; D015202:Protein Engineering; D013237:Stereoisomerism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D014867:Water / Q000378:metabolism
D010636:Phenols; D014867:Water; C011867:4-hydroxyphenylethanol; D001224:Aspartic Acid; D018698:Glutamic Acid; C042291:4-ethylphenol; D000429:Alcohol Oxidoreductases; C076865:vanillyl-alcohol oxidase; D010626:Phenylethyl Alcohol