title: Functional characterization of ent-copalyl diphosphate synthase from Andrographis paniculata with putative involvement in andrographolides biosynthesis.
abstract: OBJECTIVE
  To characterize the ent-copalyl diphosphate (ent-CPP) synthase involved in the biosynthetic pathway of andrographolides in a medicinal plant, Andrographis paniculata.
  
  
  RESULTS
  The ent-CPP synthase (ent-CPS) gene was cloned from A. paniculata and its encoded ApCPS was demonstrated to react with (E,E,E)-geranylgeranyl diphosphate to form ent-CPP through recombinant expression in Escherichia coli. Site-directed mutagenesis of the Asp to Ala in the conserved DXDD motif of ApCPS resulted in loss of function. One Arg is located in the conserved position close to DXDD motif indicating the involvement of ApCPS in specialized metabolism. In addition, RT-PCR analysis revealed that ApCPS was expressed in all tissues of A. paniculata at all growth stages, which is consistent with andrographolides accumulating in these organs. Methyl jasmonate induced ApCPS gene expression, matching inducible accumulation of andrographolides in vivo.
  
  
  CONCLUSIONS
  ApCPS is the first ent-CPS characterized in A. paniculata and is suggested to be involved in biosynthesis of andrographolides that have high pharmaceutical values.
authors.affiliation: College of Agronomy, Sichuan Agricultural University, Chengdu, 611130, China.
authors.forename: Qinqin
authors.initials: Q
authors.lastname: Shen
authors.affiliation: College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, 611130, China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Li
authors.affiliation: College of Agronomy, Sichuan Agricultural University, Chengdu, 611130, China.
authors.forename: Yu
authors.initials: Y
authors.lastname: Jiang
authors.affiliation: College of Agronomy, Sichuan Agricultural University, Chengdu, 611130, China. qwang@sicau.edu.cn.|Institute of Ecological Agriculture, Sichuan Agricultural University, Chengdu, 611130, China. qwang@sicau.edu.cn.
authors.forename: Qiang
authors.initials: Q
authors.lastname: Wang
mesh_terms: D000085:Acetates / Q000494:pharmacology; D019883:Alkyl and Aryl Transferases / Q000235:genetics* / Q000378:metabolism*; D035685:Andrographis / Q000201:enzymology* / Q000235:genetics; D003001:Cloning, Molecular; D003517:Cyclopentanes / Q000494:pharmacology; D004224:Diterpenes / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D054883:Oxylipins / Q000494:pharmacology; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism*; D014018:Tissue Distribution
chemical_list: D000085:Acetates; D003517:Cyclopentanes; D004224:Diterpenes; D054883:Oxylipins; D010940:Plant Proteins; C030419:andrographolide; C072239:methyl jasmonate; D019883:Alkyl and Aryl Transferases; C090963:ent-kaurene synthetase A
keywords: Andrographis paniculata; Andrographolides; Conserved DXDD motif; Diterpenes; Geranylgeranyl diphosphate; ent-Copalyl diphosphate synthase