This old version of Proteopedia is provided for student assignments while the new version is undergoing repairs. Content and edits done in this old version of Proteopedia after March 1, 2026 will eventually be lost when it is retired in about June of 2026.
Apply for new accounts at the new Proteopedia. Your logins will work in both the old and new versions.
1w1m
From Proteopedia
|
STRUCTURE OF THE OCTAMERIC FLAVOENZYME VANILLYL-ALCOHOL OXIDASE: GLU502GLY MUTANT
Overview
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 Structure
1W1M 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.
Reference
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
Page seeded by OCA on Mon Nov 5 17:22:25 2007
