Structure, function and redesign of vanillyl-alcohol oxidase

Abstract

Vanillyl-alcohol oxidase (VAO) from Penicillium simplicissimum is an inducible flavoprotein involved in the biodegradation of lignin-derived aromatic compounds. The enzyme is the prototype of a newly recognized family of structurally related oxidoreductases, whose members share a conserved FAD-binding domain. The flavin cofactor in VAO is covalently linked to His422 of the cap domain. This covalent interaction increases the redox potential of the flavin, facilitating the oxidation of a wide range of phenolic substrates. Covalent flavinylation is an autocatalytic process in which His61 activates His422. Vanillyl-alcohol oxidase (VAO) catalysis involves the formation of a p-quinone methide product intermediate. With 4-ethylphenol as a substrate, this intermediate is stereospecifically attacked by water, yielding the R-enantiomer of 1-(4′-hydroxyphenyl)ethanol in high enantiomeric excess. Studies from site-directed mutants revealed that Asp170 is critically involved in substrate hydroxylation. This prompted us to create a double mutation (D170S/T457E) with the idea to relocate the putative active site base to the opposite face of the substrate-binding pocket. In this way, and for the first time, the stereospecificity of a redox enzyme was inversed by rational redesign.