title: The T296V Mutant of Amorpha-4,11-diene Synthase Is Defective in Allylic Diphosphate Isomerization but Retains the Ability To Cyclize the Intermediate (3R)-Nerolidyl Diphosphate to Amorpha-4,11-diene.
abstract: The T296V mutant of amorpha-4,11-diene synthase catalyzes the abortive conversion of the natural substrate (E,E)-farnesyl diphosphate mainly into the acyclic product (E)-β-farnesene (88%) instead of the natural bicyclic sesquiterpene amorphadiene (7%). Incubation of the T296V mutant with (3R,6E)-nerolidyl diphosphate resulted in cyclization to amorphadiene. Analysis of additional mutants of amino acid residue 296 and in vitro assays with the intermediate analogue (2Z,6E)-farnesyl diphosphate as well as (3S,6E)-nerolidyl diphosphate demonstrated that the T296V mutant can no longer catalyze the allylic rearrangement of farnesyl diphosphate to the normal intermediate (3R,6E)-nerolidyl diphosphate, while retaining the ability to cyclize (3R,6E)-nerolidyl diphosphate to amorphadiene. The T296A mutant predominantly retained amorphadiene synthase activity, indicating that neither the hydroxyl nor the methyl group of the Thr296 side chain is required for cyclase activity.
authors.forename: Zhenqiu
authors.initials: Z
authors.lastname: Li
authors.forename: Ruiping
authors.initials: R
authors.lastname: Gao
authors.forename: Qinggang
authors.initials: Q
authors.lastname: Hao
authors.forename: Huifang
authors.initials: H
authors.lastname: Zhao
authors.forename: Longbin
authors.initials: L
authors.lastname: Cheng
authors.forename: Fang
authors.initials: F
authors.lastname: He
authors.forename: Li
authors.initials: L
authors.lastname: Liu
authors.forename: Xiuhua
authors.initials: X
authors.lastname: Liu
authors.affiliation: Department of Chemistry, Brown University , Box H, Providence, Rhode Island 02912-9108, United States.
authors.forename: Wayne K W
authors.initials: WK
authors.lastname: Chou
authors.forename: Huajie
authors.initials: H
authors.lastname: Zhu
authors.affiliation: Department of Chemistry, Brown University , Box H, Providence, Rhode Island 02912-9108, United States.
authors.forename: David E
authors.initials: DE
authors.lastname: Cane
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D031172:Artemisia annua / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D055162:Biocatalysis; D003500:Cyclization; D011756:Diphosphates / Q000737:chemistry* / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D020125:Mutation, Missense; D010940:Plant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D000081223:Polycyclic Sesquiterpenes; D011106:Polyisoprenyl Phosphates / Q000737:chemistry* / Q000378:metabolism; D012717:Sesquiterpenes / Q000737:chemistry* / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
chemical_list: D011756:Diphosphates; D010940:Plant Proteins; D000081223:Polycyclic Sesquiterpenes; D011106:Polyisoprenyl Phosphates; D012717:Sesquiterpenes; C515348:amorpha-4,11-diene; C004808:farnesyl pyrophosphate; D019883:Alkyl and Aryl Transferases; C403714:amorpha-4,11-diene synthase