van den Heuvel R H, Fraaije M W, Laane C, van Berkel W J
Laboratory of Biochemistry, Department of Biomolecular Sciences, Wageningen University Research Center, Wageningen, The Netherlands.
J Bacteriol. 1998 Nov;180(21):5646-51. doi: 10.1128/JB.180.21.5646-5651.1998.
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.
研究了共价黄素蛋白香草醇氧化酶对手性4-烷基苯酚的区域和立体特异性转化。该酶对带有最多七个碳原子脂肪族侧链的4-烷基苯酚具有活性。4-乙基苯酚和4-正丙基苯酚的催化效率最佳。这些短链4-烷基苯酚被立体选择性地羟基化为相应的（R）-1-（4'-羟基苯基）醇（F.P.Drijfhout、M.W.Fraaije、H.Jongejan、W.J.H.van Berkel和M.C.R.Franssen，《生物技术与生物工程》59:171 - 177，1998）。发现（S）-1-（4'-羟基苯基）乙醇是比（R）-1-（4'-羟基苯基）乙醇好得多的底物，这解释了为什么在4-乙基苯酚的酶促转化过程中几乎不形成4-羟基苯乙酮。中链4-烷基苯酚仅被香草醇氧化酶转化为相应的1-（4'-羟基苯基）烯烃。这些反应的相对顺反立体化学强烈依赖于烷基侧链的性质。4-仲丁基苯酚的酶促转化产生了两种质量相同但碎片化模式不同的（4'-羟基苯基）-仲丁烯异构体。我们得出结论，在确定香草醇氧化酶介导的4-烷基苯酚转化的区域特异性和立体化学结果时，酶活性位点的水可及性和底物疏水烷基侧链的取向至关重要。
