title: Probing the mechanism of 1,4-conjugate elimination reactions catalyzed by terpene synthases.
abstract: 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.
authors.affiliation: School of Chemistry and Cardiff Catalysis Institute, Cardiff University, Main Building, Park Place, Cardiff CF10 3AT, United Kingdom.
authors.forename: Juan A
authors.initials: JA
authors.lastname: Faraldos
authors.forename: Veronica
authors.initials: V
authors.lastname: Gonzalez
authors.forename: Amang
authors.initials: A
authors.lastname: Li
authors.forename: Fanglei
authors.initials: F
authors.lastname: Yu
authors.forename: Mustafa
authors.initials: M
authors.lastname: Köksal
authors.forename: David W
authors.initials: DW
authors.lastname: Christianson
authors.forename: Rudolf K
authors.initials: RK
authors.lastname: Allemann
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000378:metabolism*; D002070:Butadienes / Q000378:metabolism; D045782:Hemiterpenes / Q000378:metabolism; D027541:Mentha / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism; D010420:Pentanes / Q000378:metabolism; D010940:Plant Proteins / Q000378:metabolism*; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D032107:Populus / Q000378:metabolism*; D011755:Pyrophosphatases / Q000378:metabolism*; D011994:Recombinant Proteins / Q000378:metabolism; D012717:Sesquiterpenes / Q000378:metabolism
chemical_list: D002070:Butadienes; D045782:Hemiterpenes; D009943:Organophosphorus Compounds; D010420:Pentanes; D010940:Plant Proteins; D011106:Polyisoprenyl Phosphates; D011994:Recombinant Proteins; D012717:Sesquiterpenes; C005059:isoprene; C062671:beta-farnesene; C043060:3,3-dimethylallyl pyrophosphate; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C093854:isoprene synthase; C108877:terpene synthase; D011755:Pyrophosphatases; C109459:beta-farnesene synthase