Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin.

The Journal of biological chemistry (2004-06-01)
Robert H H van den Heuvel, Willy A M van den Berg, Stefano Rovida, Willem J H van Berkel
RESUMEN

The flavoenzyme vanillyl-alcohol oxidase was subjected to random mutagenesis to generate mutants with enhanced reactivity to creosol (2-methoxy-4-methylphenol). The vanillyl-alcohol oxidase-mediated conversion of creosol proceeds via a two-step process in which the initially formed vanillyl alcohol (4-hydroxy-3-methoxybenzyl alcohol) is oxidized to the widely used flavor compound vanillin (4-hydroxy-3-methoxybenzaldehyde). The first step of this reaction is extremely slow due to the formation of a covalent FAD N-5-creosol adduct. After a single round of error-prone PCR, seven mutants were generated with increased reactivity to creosol. The single-point mutants I238T, F454Y, E502G, and T505S showed an up to 40-fold increase in catalytic efficiency (kcat/Km) with creosol compared with the wild-type enzyme. This enhanced reactivity was due to a lower stability of the covalent flavin-substrate adduct, thereby promoting vanillin formation. The catalytic efficiencies of the mutants were also enhanced for other ortho-substituted 4-methylphenols, but not for p-cresol (4-methylphenol). The replaced amino acid residues are not located within a distance of direct interaction with the substrate, and the determined three-dimensional structures of the mutant enzymes are highly similar to that of the wild-type enzyme. These results clearly show the importance of remote residues, not readily predicted by rational design, for the substrate specificity of enzymes.

MATERIALES
Referencia del producto
Marca
Descripción del producto

Sigma-Aldrich
2-Methoxy-4-methylphenol, ≥98%, FG
Sigma-Aldrich
2-Methoxy-4-methylphenol, ≥98%
Sigma-Aldrich
2-Methoxy-4-methylphenol, natural, 97%, FG