title: Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin.
abstract: 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.
authors.affiliation: Department of Genetics and Microbiology, University of Pavia, via Abbiategrasso 207, 27100 Pavia, Italy.
authors.forename: Robert H H
authors.initials: RH
authors.lastname: van den Heuvel
authors.forename: Willy A M
authors.initials: WA
authors.lastname: van den Berg
authors.forename: Stefano
authors.initials: S
authors.lastname: Rovida
authors.forename: Willem J H
authors.initials: WJ
authors.lastname: van Berkel
mesh_terms: D000429:Alcohol Oxidoreductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D001547:Benzaldehydes / Q000737:chemistry / Q000378:metabolism*; D002384:Catalysis; D003408:Cresols / Q000378:metabolism; D003460:Crystallization; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005421:Flavoring Agents; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D015394:Molecular Structure; D016296:Mutagenesis; D010084:Oxidation-Reduction; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001547:Benzaldehydes; D003408:Cresols; D005421:Flavoring Agents; D011994:Recombinant Proteins; D005182:Flavin-Adenine Dinucleotide; C100058:vanillin; D000429:Alcohol Oxidoreductases; C076865:vanillyl-alcohol oxidase; C025390:creosol