REGIO-SPECIFIC AND STEREOSPECIFIC CONVERSION OF 4-ALKYLPHENOLS BY THECOVALENT FLAVOPROTEIN VANILLYL-ALCOHOL OXIDASE

The regio- and stereospecific conversion of prochiral 4-alkylphenols b y the covalent flavoprotein vanillyl-alcohol oxidase was investigated, The enzyme was active, with 4-alkylphenols bearing aliphatic side cha ins of up to seven carbon atoms. Optimal catalytic efficiency occurred with 4-ethylphenol and 4-n-propylphenols. These short-chain 4-alkylph enols are stereoselectively hydroxylated to the corresponding (R)-1-(4 '-hydroxyphenyl)alcohols (F.P. Drijfhout, M. W. Fraaije, H. Jongejan, W. J. H. van Berkel, and M. C. R. Franssen, Biotechnol. Bioeng. 59:171 -177, 1998), (S)-1-(4'-Hydroxyphenyl)ethanol was found to be a far bet ter substrate than (R)-1-(4'-hydroxyphenyl) ethanol, explaining why du ring the enzymatic conversion of 4-ethylphenol nearly no 4-hydroxyacet ophenone is formed. Medium-chain 4-alkylphenols were exclusively conve rted by vanillyl-alcohol oxidase to the corresponding 1-(4'-hydroxyphe nyl)alkenes. The relative cis-trans stereochemistry of these reactions was strongly dependent on the nature of the alkyl side chain, The enz ymatic conversion of 4-sec-butylphenol resulted in two (4'-hydroxyphen yl)-sec-butene isomers with identical masses but different fragmentati on patterns. We conclude that the water accessibility of the enzyme ac tive site and the orientation of the hydrophobic alkyl side chain of t he substrate are of major importance in determining the regiospecific and stereochemical outcome of vanillyl-alcohol oxidase-mediated conver sions of 4-alkylphenols.