1W1K

STRUCTURE OF THE OCTAMERIC FLAVOENZYME VANILLYL-ALCOHOL OXIDASE: Ile238Thr Mutant

Summary for 1W1K

• Entry DOI: 10.2210/pdb1w1k/pdb
• Related: 1AHU 1AHV 1AHZ 1DZN 1E0Y 1E8F 1E8G 1E8H 1QLT 1QLU 1VAO 1W1J 1W1L 1W1M 2VAO
• Descriptor: VANILLYL-ALCOHOL OXIDASE, FLAVIN-ADENINE DINUCLEOTIDE, 2-methoxy-4-[(1E)-prop-1-en-1-yl]phenol (3 entities in total)
• Functional Keywords: flavoenzyme, oxidoreductase, catalysis, fad
• Biological source: PENICILLIUM SIMPLICISSIMUM
• Cellular location: Peroxisome: P56216
• Total number of polymer chains: 2
• Total formula weight: 128089.63

Authors

Van Den Heuvel, R.H. (deposition date: 2004-06-22, release date: 2004-07-02, Last modification date: 2024-11-20)

Primary citation

Van Den Heuvel, R.H.,Van Den Berg, W.A.,Rovida, S.,Van Berkel, W.J.
Laboratory-Evolved Vanillyl-Alcohol Oxidase Produces Natural Vanillin
J.Biol.Chem., 279:33492-, 2004

PubMed 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.

PubMed: 15169773
DOI: 10.1074/JBC.M312968200

Experimental method

X-RAY DIFFRACTION (2.55 Å)