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Journal of Bacteriology, November 1998, p. 5646-5651, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.

Regio- and Stereospecific Conversion of 4-Alkylphenols by the Covalent Flavoprotein Vanillyl-Alcohol Oxidase

Robert H. H. van den Heuvel, Marco W. Fraaije, Colja Laane, and Willem J. H. van Berkel*

Laboratory of Biochemistry, Department of Biomolecular Sciences, Wageningen University Research Center, Wageningen, The Netherlands

Received 22 June 1998/Accepted 31 August 1998

The regio- and stereospecific conversion of prochiral 4-alkylphenols by the covalent flavoprotein vanillyl-alcohol oxidase was investigated. The enzyme was active, with 4-alkylphenols bearing aliphatic side chains of up to seven carbon atoms. Optimal catalytic efficiency occurred with 4-ethylphenol and 4-n-propylphenols. These short-chain 4-alkylphenols 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 better substrate than (R)-1-(4'-hydroxyphenyl)ethanol, explaining why during the enzymatic conversion of 4-ethylphenol nearly no 4-hydroxyacetophenone is formed. Medium-chain 4-alkylphenols were exclusively converted by vanillyl-alcohol oxidase to the corresponding 1-(4'-hydroxyphenyl)alkenes. The relative cis-trans stereochemistry of these reactions was strongly dependent on the nature of the alkyl side chain. The enzymatic conversion of 4-sec-butylphenol resulted in two (4'-hydroxyphenyl)-sec-butene isomers with identical masses but different fragmentation patterns. We conclude that the water accessibility of the enzyme active site and the orientation of the hydrophobic alkyl side chain of the substrate are of major importance in determining the regiospecific and stereochemical outcome of vanillyl-alcohol oxidase-mediated conversions of 4-alkylphenols.

* Corresponding author. Mailing address: Department of Biomolecular Sciences, Laboratory of Biochemistry, Wageningen University Research Center, Dreijenlaan 3, 6703 HA Wageningen, The Netherlands. Phone: 31-317-482861. Fax: 31-317-484801. E-mail: willem.vanberkel{at}fad.bc.wau.nl.

Journal of Bacteriology, November 1998, p. 5646-5651, Vol. 180, No. 21
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.


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