Exploring the biocatalytic potential of vanillyl-alcohol oxidase by site-directed mutagenesis

Vanillyl-alcohol oxidase (VAO) is a flavoprotein containing a covalently bo und FAD cofactor. The enzyme acts on a wide variety of 4-alkylphenols beari ng aliphatic side-chains up to seven carbon atoms in length. Short-chain 4- alkylphenols are predominantly hydroxylated to (R)-1-(4'-hydroxyphenyl) alc ohols, whereas medium-chain 4-alkylphenols are dehydrogenated to the corres ponding I(4'-hydroxyphenyl)alkenes. In this account, we summarize our work on the structure, mechanism, and biocatalytic potential of VAO. It is shown that the efficiency of hydroxylation of 4-alkylphenols is dependent on the type or amino acid residue engineered at position 170. Furthermore, it is demonstrated that the stereospecificity of the hydroxylation reaction can b e inverted by relocating the active site base to the opposite face or the s ubstrate-binding pocket.