title: Heteromeric and homomeric geranyl diphosphate synthases from Catharanthus roseus and their role in monoterpene indole alkaloid biosynthesis.
abstract: Catharanthus roseus is the sole source of two most important monoterpene indole alkaloid (MIA) anti-cancer agents: vinblastine and vincristine. MIAs possess a terpene and an indole moiety derived from terpenoid and shikimate pathways, respectively. Geranyl diphosphate (GPP), the entry point to the formation of terpene moiety, is a product of the condensation of isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) by GPP synthase (GPPS). Here, we report three genes encoding proteins with sequence similarity to large subunit (CrGPPS.LSU) and small subunit (CrGPPS.SSU) of heteromeric GPPSs, and a homomeric GPPSs. CrGPPS.LSU is a bifunctional enzyme producing both GPP and geranyl geranyl diphosphate (GGPP), CrGPPS.SSU is inactive, whereas CrGPPS is a homomeric enzyme forming GPP. Co-expression of both subunits in Escherichia coli resulted in heteromeric enzyme with enhanced activity producing only GPP. While CrGPPS.LSU and CrGPPS showed higher expression in older and younger leaves, respectively, CrGPPS.SSU showed an increasing trend and decreased gradually. Methyl jasmonate (MeJA) treatment of leaves significantly induced the expression of only CrGPPS.SSU. GFP localization indicated that CrGPPS.SSU is plastidial whereas CrGPPS is mitochondrial. Transient overexpression of AmGPPS.SSU in C. roseus leaves resulted in increased vindoline, immediate monomeric precursor of vinblastine and vincristine. Although C. roseus has both heteromeric and homomeric GPPS enzymes, our results implicate the involvement of only heteromeric GPPS with CrGPPS.SSU regulating the GPP flux for MIA biosynthesis.
authors.affiliation: CSIR-Central Institute of Medicinal and Aromatic Plants Research Centre, Bangalore, 560065, India.
authors.forename: Avanish
authors.initials: A
authors.lastname: Rai
authors.forename: Shachi S
authors.initials: SS
authors.lastname: Smita
authors.forename: Anup Kumar
authors.initials: AK
authors.lastname: Singh
authors.forename: Karuna
authors.initials: K
authors.lastname: Shanker
authors.forename: Dinesh A
authors.initials: DA
authors.lastname: Nagegowda
mesh_terms: D019883:Alkyl and Aryl Transferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D053898:Biosynthetic Pathways / Q000235:genetics; D030008:Catharanthus / Q000201:enzymology*; D018076:DNA, Complementary / Q000235:genetics / Q000302:isolation & purification; D011756:Diphosphates / Q000378:metabolism*; D004224:Diterpenes / Q000378:metabolism*; D057075:Enzyme Assays; D004926:Escherichia coli / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D005816:Genetic Complementation Test; D026121:Indole Alkaloids / Q000378:metabolism*; D008969:Molecular Sequence Data; D039821:Monoterpenes / Q000378:metabolism*; D010802:Phylogeny; D018515:Plant Leaves / Q000235:genetics / Q000254:growth & development; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055503:Protein Multimerization; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D062211:Spatio-Temporal Analysis; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D018076:DNA, Complementary; D011756:Diphosphates; D004224:Diterpenes; D026121:Indole Alkaloids; D039821:Monoterpenes; D010940:Plant Proteins; D011994:Recombinant Proteins; C511282:geranyl diphosphate; D019883:Alkyl and Aryl Transferases
keywords: C. roseus; geranyl diphosphate synthase; heteromer; homomer; large subunit; monoterpene indole alkaloids; small subunit; vindoline.