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Exploring the Biocatalytic Potential of Vanillyl‐Alcohol Oxidase by Site‐Directed Mutagenesis
Author(s) -
Heuvel Robert H. H. van den,
Laane Colja,
Berkel Willem J. H. van
Publication year - 2001
Publication title -
advanced synthesis and catalysis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.541
H-Index - 155
eISSN - 1615-4169
pISSN - 1615-4150
DOI - 10.1002/1615-4169(200108)343:6/7<515::aid-adsc515>3.0.co;2-u
Subject(s) - chemistry , site directed mutagenesis , mutagenesis , alcohol , alcohol oxidase , organic chemistry , biochemistry , mutation , recombinant dna , gene , pichia pastoris , mutant
Vanillyl‐alcohol oxidase (VAO) is a flavoprotein containing a covalently bound FAD cofactor. The enzyme acts on a wide variety of 4‐alkylphenols bearing aliphatic side‐chains up to seven carbon atoms in length. Short‐chain 4‐alkylphenols are predominantly hydroxylated to ( R )‐1‐(4’‐hydroxyphenyl) alcohols, whereas medium‐chain 4‐alkylphenols are dehydrogenated to the corresponding 1‐(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 of amino acid residue engineered at position 170. Furthermore, it is demonstrated that the stereospecificity of the hydroxylation reaction can be inverted by relocating the active site base to the opposite face of the substrate‐binding pocket. 1 Introduction 2 Vanillyl‐Alcohol Oxidase 3 Structural Properties of Vanillyl‐Alcohol Oxidase 4 Catalytic and Structural Properties of Asp170 Variants 5 Efficiency of Hydroxylation of 4‐Alkylphenols 6 Stereospecificity of Hydroxylation 7 Conclusion