title: A new approach for the investigation of isoprenoid biosynthesis featuring pathway switching, deuterium hyperlabeling, and 1H NMR spectroscopy. The reaction mechanism of a novel streptomyces diterpene cyclase.
abstract: Recent methodology for the investigation of isoprenoid biosynthesis featuring pathway switching and hyperdeuteration has shown significant advantages in elucidating the reaction mechanism of a novel Streptomyces diterpene cyclase with use of precise atom-level analysis. Insight into the cyclization mechanism involved in the conversion of geranylgeranyl diphosphate (GGPP) into a clerodane hydrocarbon terpentetriene was obtained by heterologous expression in doubly engineered Streptomyces lividans of a diterpene cyclase gene derived from Streptomyces griseolosporeus, a producer of an unique diterpenoid cytotoxic antibiotic terpentecin, and by in vivo labeling with mevalonate-d(9). The cyclization involved electrophilic protonation, cationic ring closure, Wagner-Meerwein-type rearrangements, and deprotonation. A key feature was that the labeled metabolite as a mixture of predominantly deuterated mosaic molecules provided sufficient information that close analysis of the labeling pattern for each individual isoprene unit was achieved primarily by (1)H NMR spectroscopy. The cyclization of GGPP into the clerodane skeleton catalyzed by the cyclase appears to involve Si-face specific protonation, intermediates with A/B chair-boat conformation, and specific methyl and hydride migrations to give an intermediary C-4 carbocation. Subsequent collapse of the cation through specific removal of the initiating proton and final elimination of diphosphate gives rise to the terpentetriene hydrocarbon.
authors.affiliation: Department of Chemistry and Materials Science, Tokyo Institute of Technology, O-okayama, Meguro-ku, Tokyo 152-8551, Japan. eguchi@cms.titech.ac.jp
authors.forename: Tadashi
authors.initials: T
authors.lastname: Eguchi
authors.forename: Yasumasa
authors.initials: Y
authors.lastname: Dekishima
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Hamano
authors.forename: Tohru
authors.initials: T
authors.lastname: Dairi
authors.forename: Haruo
authors.initials: H
authors.lastname: Seto
authors.forename: Katsumi
authors.initials: K
authors.lastname: Kakinuma
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism*; D003500:Cyclization; D003903:Deuterium; D004224:Diterpenes / Q000378:metabolism; D019753:Intramolecular Lyases / Q000737:chemistry / Q000378:metabolism*; D007553:Isotope Labeling; D009682:Magnetic Resonance Spectroscopy; D015394:Molecular Structure; D011106:Polyisoprenyl Phosphates / Q000378:metabolism; D013302:Streptomyces / Q000201:enzymology*; D013729:Terpenes / Q000378:metabolism*
chemical_list: D001426:Bacterial Proteins; D004224:Diterpenes; D011106:Polyisoprenyl Phosphates; D013729:Terpenes; D003903:Deuterium; D019753:Intramolecular Lyases; C002963:geranylgeranyl pyrophosphate