Inversion of stereospecificity of vanillyl-alcohol oxidase

Vanillyl-alcohol oxidase (VAO) is the prototype of a newly recognized famil y of structurally related oxidoreductases sharing a conserved FAD-binding d omain. The active site of VAO is formed by a cavity where the enzyme is abl e to catalyze many reactions with phenolic substrates. Among these reaction s is the stereospecific hydroxylation of 4-ethylphenol-forming (R)-1(4'-hyd roxyphenyl)ethanol. During this conversion, Asp-170 is probably critical fo r 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 s tereospecificity has been achieved. Like native VAO, the single mutants T45 7E, D170A, and D170S preferentially converted 4-ethylphenol to the (R)-enan tiomer of 1-(4'-hydroxyphenyl)ethanol. The double mutants D170A/T457E and D 170S/T457E exhibited an inverted stereospecificity with 4-ethylphenol. Part icularly, D1705/T457E was strongly (S)-selective, with an enantiomeric exce ss of 80%. The crystal structure of D170S/T457E, in complex with trifluorom ethylphenol, 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.