1w1k
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STRUCTURE OF THE OCTAMERIC FLAVOENZYME VANILLYL-ALCOHOL OXIDASE: ILE238THR MUTANT
OverviewOverview
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.
About this StructureAbout this Structure
1W1K is a Single protein structure of sequence from Penicillium simplicissimum with FAD and EUG as ligands. Active as Alcohol oxidase, with EC number 1.1.3.13 Structure known Active Site: AC1. Full crystallographic information is available from OCA.
ReferenceReference
Laboratory-evolved vanillyl-alcohol oxidase produces natural vanillin., van den Heuvel RH, van den Berg WA, Rovida S, van Berkel WJ, J Biol Chem. 2004 Aug 6;279(32):33492-500. Epub 2004 May 28. PMID:15169773
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