title: Functional characterization of NES and GES responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool in Tripterygium wilfordii.
abstract: Triptolide and celastrol, two principal bioactive compounds in Tripterygium wilfordii, are produced from geranylgeranyl diphosphate (GGPP) and farnesyl diphosphate ((E,E)-FPP) through terpenoid biosynthesis pathway. However, little is known about T. wilfordii terpene synthases which could competitively utilize GGPP and (E,E)-FPP as substrates, producing C15and C20tertiary alcohols. Here we firstly cloned the genes encoding nerolidol synthase (NES) and geranyllinalool synthases (GES1, GES2), which are responsible for the biosynthesis of (E)-nerolidol and (E,E)-geranyllinalool. In vitro characterization of recombinant TwNES and TwGES1 revealed both were functional enzymes that could catalyze the conversion of (E,E)-FPP and GGPP to (E)-nerolidol and (E,E)-geranyllinalool, which were consistent with the results of yeast fermentation. Biochemical characterization revealed TwNES and TwGES1 had strong dependency for Mg2+, Kmand Kcat/Kmvalues of TwNES for (E,E)-FPP were 12.700 μM and 0.029 s-1/μM, and TwGES1 for GGPP were 2.039 μM and 0.019 s-1/μM. Real-time PCR analysis showed the expression levels of NES and GES1 increased by several fold in the suspension cells treated with alamethicin, indicating TwNES and TwGES1 are likely to utilize GGPP and (E,E)-FPP to generate tertiary alcohols as precursor of plant volatiles, which play important roles in the ecological interactions between T. wilfordii and other organisms.
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.|State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
authors.forename: Ping
authors.initials: P
authors.lastname: Su
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.
authors.forename: Tianyuan
authors.initials: T
authors.lastname: Hu
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.
authors.forename: Yujia
authors.initials: Y
authors.lastname: Liu
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.|State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
authors.forename: Yuru
authors.initials: Y
authors.lastname: Tong
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.|State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
authors.forename: Hongyu
authors.initials: H
authors.lastname: Guan
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.|State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
authors.forename: Yifeng
authors.initials: Y
authors.lastname: Zhang
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.
authors.forename: Jiawei
authors.initials: J
authors.lastname: Zhou
authors.affiliation: State Key Laboratory Breeding Base of Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing, China.
authors.forename: Luqi
authors.initials: L
authors.lastname: Huang
authors.affiliation: School of Traditional Chinese Medicine, Capital Medical University, Beijing, China.|Beijing Key Lab of TCM Collateral Disease Theory Research, Beijing, China.
authors.forename: Wei
authors.initials: W
authors.lastname: Gao
mesh_terms: D000080462:Acyclic Monoterpenes; D003067:Coenzymes / Q000378:metabolism; D004224:Diterpenes / Q000378:metabolism*; D008274:Magnesium / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010946:Plants, Medicinal / Q000201:enzymology / Q000235:genetics / Q000378:metabolism; D012717:Sesquiterpenes / Q000378:metabolism*; D013379:Substrate Specificity; D014166:Transferases / Q000235:genetics* / Q000378:metabolism; D029757:Tripterygium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism
chemical_list: D000080462:Acyclic Monoterpenes; D003067:Coenzymes; D004224:Diterpenes; D010940:Plant Proteins; D012717:Sesquiterpenes; D014166:Transferases; D008274:Magnesium; C037055:nerolidol; C534312:3,7,11,15-tetramethyl-1,6,10,14-hexadecatetraen-3-ol