Probing the Mechanism of 1,4-Conjugate Elimination Reactions Catalyzed by Terpene Synthases

• Published in: Journal of the American Chemical Society Vol. 134; no. 51; pp. 20844 - 20848
• Main Authors: Faraldos, Juan A, Gonzalez, Veronica, Li, Amang, Yu, Fanglei, Köksal, Mustafa, Christianson, David W, Allemann, Rudolf K
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 26.12.2012
• Subjects: Alkyl and Aryl Transferases - metabolism; Butadienes - metabolism; Hemiterpenes - metabolism; Mentha - metabolism; Organophosphorus Compounds - metabolism; Pentanes - metabolism; Plant Proteins - metabolism; Polyisoprenyl Phosphates - metabolism; Populus - metabolism; Pyrophosphatases - metabolism; Recombinant Proteins - metabolism; Sesquiterpenes - metabolism
• ISSN: 0002-7863; 1520-5126
• DOI: 10.1021/ja311022s

The reaction mechanisms of (E)-β-farnesene synthase (EBFS) and isoprene synthase (ISPS), enzymes that catalyze a formal regiospecific 1,4-conjugate elimination of hydrogen diphosphate from (E,E)-farnesyl and dimethylallyl diphosphate (FDP and DMADP) to generate the semiochemicals (E)-β-farnesene and isoprene, respectively, were probed with substrate analogs and kinetic measurements. The results support stepwise reaction mechanisms through analogous enzyme-bound allylic cationic intermediates. For EBFS, we demonstrate that the elimination reaction can proceed via the enzyme-bound intermediate trans-nerolidyl diphosphate, while for ISPS the intermediacy of 2-methylbut-3-enyl 2-diphosphate can be inferred from the product outcome when deuterated DMADPs are used as substrates. Possible implications derived from the mechanistic details of the EBFS-catalyzed reaction for the evolution of sesquiterpene synthases are discussed.