title: Functional expression of a putative geraniol 8-hydroxylase by reconstitution of bacterially expressed plant CYP76F45 and NADPH-cytochrome P450 reductase CPR I from Croton stellatopilosus Ohba.
abstract: While attempting to isolate the enzyme geranylgeraniol 18-hydroxylase, which is involved in plaunotol biosynthesis in Croton stellatopilosus (Cs), the cDNAs for a cytochrome P450 monooxygenase(designated as CYP76F45) and an NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from the leaf. The CYP76F45 and CsCPR I genes have open reading frames (ORFs) encoding 507- and 711-amino acid proteins with predicted relative molecular weights of 56.7 and 79.0 kDa,respectively. Amino acid sequence comparison showed that both CYP76F45 (63–73%) and CsCPR I (79–83%) share relatively high sequence identities with homologous proteins in other plant species.Phylogenetic tree analysis confirmed that CYP76F45 belongs to the CYP76 family and that CsCPR I belongs to Class I of dicotyledonous CPRs, with both being closely related to Ricinus communis genes. Functional characterization of both enzymes, each expressed separately in Escherichia coli as recombinant proteins,showed that only simultaneous incubation of the membrane bound proteins with the substrate geraniol (GOH) and the coenzyme NADPH could form 8-hydroxygeraniol. The enzyme mixture could also utilize acyclic sesquiterpene farnesol (FOH) with a comparable substrate preference ratio (GOH:FOH) of 54:46. The levelsof the CYP76F45 and CsCPR I transcripts in the shoots, leaves and twigs of C. stellatopilosus were correlated with the levels of a major monoterpenoid indole alkaloid, identified tentatively as 19-Evallesamine,that accumulated in these plant parts. These results suggested that CYP76F45 and CPR I function as the enzyme geraniol-8-hydroxylase (G8H), which is likely to be involved in the biosynthesis of the indole alkaloid in C. stellatopilosus [corrected].
authors: Program in Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand; Research Unit for Natural Product Biotechnology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand.
authors: Siriluk
authors: S
authors: Sintupachee
authors: Research Unit for Natural Product Biotechnology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; Division of General Science, Faculty of Education, Buriram Rajabhat University, Buriram 31000, Thailand.
authors: Worrawat
authors: W
authors: Promden
authors: Department of Chemistry, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand.
authors: Nattaya
authors: N
authors: Ngamrojanavanich
authors: Department of Pharmacognosy, Faculty of Pharmacy, Srinakharinwirot University, Nakonnayok 26120, Thailand.
authors: Worapan
authors: W
authors: Sitthithaworn
authors: Research Unit for Natural Product Biotechnology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; Department of Pharmacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: dwanchai@chula.ac.th.
authors: Wanchai
authors: W
authors: De-Eknamkul
mesh_terms: D000080462:Acyclic Monoterpenes; D000595:Amino Acid Sequence; D029802:Croton / Q000201:enzymology* / Q000235:genetics; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism*; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D009251:NADPH-Ferrihemoprotein Reductase / Q000378:metabolism*; D010802:Phylogeny; D018515:Plant Leaves / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013729:Terpenes / Q000032:analysis
chemical_list: C471578:8-hydroxygeraniol; D000080462:Acyclic Monoterpenes; D018076:DNA, Complementary; D011994:Recombinant Proteins; D013729:Terpenes; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C007836:geraniol
keywords: 19-E-vallesamine; CYP76F45; Croton stellatopilosus Ohba; Euphorbiaceae; Functional expression; Geraniol-8-hydroxylase; Monoterpenoid indole alkaloid; P450 reductase type I