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.
van Den Heuvel
R H
RH
Department of Biomolecular Sciences, Laboratory of Biochemistry, Wageningen University, The Netherlands.
Fraaije
M W
MW
Ferrer
M
M
Mattevi
A
A
van Berkel
W J
WJ
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