1W1J

STRUCTURE OF THE OCTAMERIC FLAVOENZYME VANILLYL-ALCOHOL OXIDASE: The505Ser Mutant

1W1J の概要

• エントリーDOI: 10.2210/pdb1w1j/pdb
• 関連するPDBエントリー: 1AHU 1AHV 1AHZ 1DZN 1E0Y 1E8F 1E8G 1E8H 1QLT 1QLU 1VAO 1W1K 1W1L 1W1M 2VAO
• 分子名称: VANILLYL-ALCOHOL OXIDASE, FLAVIN-ADENINE DINUCLEOTIDE, 2-methoxy-4-[(1E)-prop-1-en-1-yl]phenol (3 entities in total)
• 機能のキーワード: flavoenzyme, oxidoreductase, catalysis, fad
• 由来する生物種: PENICILLIUM SIMPLICISSIMUM
• 細胞内の位置: Peroxisome: P56216
• タンパク質・核酸の鎖数: 2
• 化学式量合計: 128085.70

構造登録者

Van Den Heuvel, R.H. (登録日: 2004-06-22, 公開日: 2004-07-02, 最終更新日: 2024-11-13)

主引用文献

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

実験手法

X-RAY DIFFRACTION (2.7 Å)