title: Molecular cloning, bacterial expression and functional characterisation of cytochrome P450 monooxygenase, CYP97C27, and NADPH-cytochrome P450 reductase, CPR I, from Croton stellatopilosus Ohba.
abstract: The cDNAs for cytochrome P450 monooxygenase (designated as CYP97C27 by D. Nelson's group) and NADPH-cytochrome P450 reductase (designated as CPR I based on its classification) were isolated from Croton stellatopilosus leaves, which actively biosynthesise plaunotol (18-OH geranylgeraniol). CYP97C27 and CPR I contain open reading frames encoding proteins of 471 and 711 amino acids with predicted molecular masses of 53 and 79kDa, respectively. By aligning the deduced sequences of CYP97C27 and CPR I with other plant species, all functional domains of CYP97C27 (heme and oxygen binding) and CPR I (CYP- and FMN, FAD, and NADPH cofactor binding) were identified. Amino acid sequence comparison indicated that both CYP97C27 (85-93%) and CPR I (79-83%) share high sequence identities with homologous proteins in other plant species, suggesting that CYP97C27 belongs to the CYP97C subfamily and that CPR I belongs to class I of the dicotyledonous CPR. Functional characterisation of both enzymes, produced in Escherichia coli (pET32a/BL21(DE3)) as recombinant proteins, showed that simultaneous incubation of CYP97C27 and CPR I with the substrate geranylgeraniol (GGOH) and coenzyme NADPH led to formation of the product plaunotol. In C. stellatopilosus, the levels of the CYP97C27 and CPR I transcripts were highly correlated with those of several mRNAs involved in the plaunotol biosynthetic pathway, suggesting that CYP97C27 and CPR I are the enzymes that catalyse the last hydroxylation step of the pathway.
authors.affiliation: 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.forename: Siriluk
authors.initials: S
authors.lastname: Sintupachee
authors.affiliation: Department of Chemistry, Faculty of Science, Chulalongkorn University Bangkok 10330, Thailand.
authors.forename: Nattaya
authors.initials: N
authors.lastname: Ngamrojanavanich
authors.affiliation: Department of Pharmacognosy, Faculty of Pharmacy, Srinakharinwirot University, Nakonnayok 26120, Thailand.
authors.forename: Worapan
authors.initials: W
authors.lastname: Sitthithaworn
authors.affiliation: Research Unit for Natural Product Biotechnology, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand; Department of Phamacognosy and Pharmaceutical Botany, Faculty of Pharmaceutical Sciences, Chulalongkorn University, Bangkok 10330, Thailand. Electronic address: dwanchai@chula.ac.th.
authors.forename: Wanchai
authors.initials: W
authors.lastname: De-Eknamkul
mesh_terms: D000595:Amino Acid Sequence; D053898:Biosynthetic Pathways; D002855:Chromatography, Thin Layer; D003001:Cloning, Molecular; D029802:Croton / Q000201:enzymology* / Q000235:genetics*; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D004224:Diterpenes; D004926:Escherichia coli / Q000378:metabolism*; D005233:Fatty Alcohols / Q000737:chemistry / Q000378:metabolism; D020869:Gene Expression Profiling; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D016415:Sequence Alignment
chemical_list: D004224:Diterpenes; D005233:Fatty Alcohols; D012333:RNA, Messenger; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D009251:NADPH-Ferrihemoprotein Reductase; C047686:plaunotol
keywords: Croton stellatopilosus Ohba; Cytochrome P450 monooxygenase; Functional characterisation; Gene cloning; NADPH-cytochrome P450 reductase; Plaunotol biosynthetic pathway