1W1M: Structure Of The Octameric Flavoenzyme Vanillyl-alcohol Oxidase: Glu502gly Mutant

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.
PDB ID: 1W1MDownload
MMDB ID: 28833
PDB Deposition Date: 2004/6/22
Updated in MMDB: 2007/10
Experimental Method:
x-ray diffraction
Resolution: 3  Å
Source Organism:
Similar Structures:
Biological Unit for 1W1M: dimeric; determined by author and by software (PQS)
Molecular Components in 1W1M
Label Count Molecule
Proteins (2 molecules)
Vanillyl-alcohol Oxidase
Molecule annotation
Chemicals (4 molecules)
* Click molecule labels to explore molecular sequence information.

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