Strictly Conserved Residues in Euphorbia tirucalli -Amyrin Cyclase: Trp612 Stabilizes Transient Cation through Cation- Interaction and CH- Interaction of Tyr736 with Leu734 Confers Robust Local Protein Architecture

作者：Aiba Yukari; Watanabe Takumi; Terasawa Yuri; Nakano Chiaki; Hoshino Tsutomu^*

来源：ChemBioChem, 2018, 19(5): 486-495.

DOI：10.1002/cbic.201700572

• biosynthesis
• molecular modeling
• mutagenesis
• proteins
• terpenoids

The functions of Trp612, Leu734, and Tyr736 of Euphorbia tirucalli -amyrin synthase were examined. The aliphatic variants (Ala, Val, Met) of Trp612 showed almost no activity, but the aromatic variants exhibited high activities: 12.5% of the wild-type activity for the W612H variant, 43% for W612F, and 63% for W612Y. That is, the enzymatic activities of the variants increased in proportion to the increase in -electron density. Thus, the major function of Trp612 is to stabilize transient cations through a cation- interaction. The Phe and Tyr variants caused a distorted folding conformation, especially at the E-ring site, which generated the aberrantly cyclized products germanicol and lupeol. The L734G and L734A variants exhibited significantly decreased activities but yielded taraxerol in a high production ratio. The Val, Ile, and Met variants showed markedly high activities (56-78% of wild-type activity); therefore, appropriate steric bulk is required at this position. The aliphatic variants of Tyr736 showed markedly decreased activities, but the Phe mutant exhibited high activity (67%), which indicates that the electrons are critical for catalysis. Homology modeling indicated that Tyr736 and Leu734 are perpendicular to the substrate and are situated face to face, which suggests that a CH- interaction occurs between Tyr736 and Leu734, reinforcing the protein architecture, and that Tyr736 cannot stabilize cationic intermediates through a cation- interaction.

• 出版日期2018-3-2