title: Structural elucidation of cisoid and transoid cyclization pathways of a sesquiterpene synthase using 2-fluorofarnesyl diphosphates.
abstract: Sesquiterpene skeletal complexity in nature originates from the enzyme-catalyzed ionization of (trans,trans)-farnesyl diphosphate (FPP) (1a) and subsequent cyclization along either 2,3-transoid or 2,3-cisoid farnesyl cation pathways. Tobacco 5-epi-aristolochene synthase (TEAS), a transoid synthase, produces cisoid products as a component of its minor product spectrum. To investigate the cryptic cisoid cyclization pathway in TEAS, we employed (cis,trans)-FPP (1b) as an alternative substrate. Strikingly, TEAS was catalytically robust in the enzymatic conversion of (cis,trans)-FPP (1b) to exclusively (>/=99.5%) cisoid products. Further, crystallographic characterization of wild-type TEAS and a catalytically promiscuous mutant (M4 TEAS) with 2-fluoro analogues of both all-trans FPP (1a) and (cis,trans)-FPP (1b) revealed binding modes consistent with preorganization of the farnesyl chain. These results provide a structural glimpse into both cisoid and transoid cyclization pathways efficiently templated by a single enzyme active site, consistent with the recently elucidated stereochemistry of the cisoid products. Further, computational studies using density functional theory calculations reveal concerted, highly asynchronous cyclization pathways leading to the major cisoid cyclization products. The implications of these discoveries for expanded sesquiterpene diversity in nature are discussed.
authors.affiliation: Howard Hughes Medical Institute, USA.
authors.forename: Joseph P
authors.initials: JP
authors.lastname: Noel
authors.forename: Nikki
authors.initials: N
authors.lastname: Dellas
authors.forename: Juan A
authors.initials: JA
authors.lastname: Faraldos
authors.forename: Marylin
authors.initials: M
authors.lastname: Zhao
authors.forename: B Andes
authors.initials: BA
authors.lastname: Hess
authors.forename: Lidia
authors.initials: L
authors.lastname: Smentek
authors.forename: Robert M
authors.initials: RM
authors.lastname: Coates
authors.forename: Paul E
authors.initials: PE
authors.lastname: O'Maille
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D018360:Crystallography, X-Ray; D003500:Cyclization; D008958:Models, Molecular; D009154:Mutation; D011106:Polyisoprenyl Phosphates / Q000737:chemistry* / Q000378:metabolism*; D012717:Sesquiterpenes / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity; D014026:Nicotiana / Q000201:enzymology*
chemical_list: C525013:2-fluorofarnesyl diphosphate; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; D019883:Alkyl and Aryl Transferases; C078217:5-epi-aristolochene synthase