title: Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant.
abstract: Camptothecin (CAM), a complex pentacyclic pyrroloqinoline alkaloid, is the starting material for CAM-type drugs that are well-known antitumor plant drugs. Although many chemical and biological research efforts have been performed to produce CAM, a few attempts have been made to uncover the enzymatic mechanism involved in the biosynthesis of CAM. Enzyme-catalyzed oxidoreduction reactions are ubiquitously presented in living organisms, especially in the biosynthetic pathway of most secondary metabolites such as CAM. Due to a lack of its reduction partner, most catalytic oxidation steps involved in the biosynthesis of CAM have not been established. In the present study, an NADPH-cytochrome P450 reductase (CPR) encoding gene CamCPR was cloned from Camptotheca acuminata, a CAM-producing plant. The full length of CamCPR cDNA contained an open reading frame of 2127-bp nucleotides, corresponding to 708-amino acid residues. CamCPR showed 70 ~ 85% identities to other characterized plant CPRs and it was categorized to the group II of CPRs on the basis of the results of multiple sequence alignment of the N-terminal hydrophobic regions. The intact and truncate CamCPRs with N- or C-terminal His6-tag were heterologously overexpressed in Escherichia coli. The recombinant enzymes showed NADPH-dependent reductase activity toward a chemical substrate ferricyanide and a protein substrate cytochrome c. The N-terminal His6-tagged CamCPR showed 18- ~ 30-fold reduction activity higher than the C-terminal His6-tagged CamCPR, which supported a reported conclusion, i.e., the last C-terminal tryptophan of CPRs plays an important role in the discrimination between NADPH and NADH. Co-expression of CamCPR and a P450 monooxygenase, CYP73A25, a cinnamate 4-hydroxylase from cotton, and the following catalytic formation of p-coumaric acid suggested that CamCPR transforms electrons from NADPH to the heme center of P450 to support its oxidation reaction. Quantitative real-time PCR analysis showed that CamCPR was expressed in the roots, stems, and leaves of C. acuminata seedlings. The relative transcript level of CamCPR in leaves was 2.2-fold higher than that of roots and the stems showed 1.5-fold transcript level higher than the roots. The functional characterization of CamCPR will be helpful to disclose the mysterious mechanisms of the biosynthesis of CAM. The present study established a platform to characterize the P450 enzymes involved in the growth, development, and metabolism of eukaryotic organisms.
authors.affiliation: Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, PR China; University of Chinese Academy of Sciences, Beijing, PR China.
authors.forename: Xixing
authors.initials: X
authors.lastname: Qu
authors.affiliation: Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, PR China; University of Chinese Academy of Sciences, Beijing, PR China.
authors.forename: Xiang
authors.initials: X
authors.lastname: Pu
authors.affiliation: Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, PR China; University of Chinese Academy of Sciences, Beijing, PR China.
authors.forename: Fei
authors.initials: F
authors.lastname: Chen
authors.affiliation: Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, PR China; University of Chinese Academy of Sciences, Beijing, PR China.
authors.forename: Yun
authors.initials: Y
authors.lastname: Yang
authors.affiliation: Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, PR China; University of Chinese Academy of Sciences, Beijing, PR China.
authors.forename: Lixia
authors.initials: L
authors.lastname: Yang
authors.affiliation: Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, PR China.
authors.forename: Guolin
authors.initials: G
authors.lastname: Zhang
authors.affiliation: Center for Natural Products Research, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu, PR China; State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai, PR China.
authors.forename: Yinggang
authors.initials: Y
authors.lastname: Luo
mesh_terms: D000595:Amino Acid Sequence; D000970:Antineoplastic Agents / Q000737:chemistry; D029769:Camptotheca / Q000201:enzymology / Q000235:genetics*; D002166:Camptothecin / Q000096:biosynthesis*; D002384:Catalysis; D003001:Cloning, Molecular; D045304:Cytochromes c / Q000737:chemistry; D018076:DNA, Complementary / Q000235:genetics; D004926:Escherichia coli / Q000378:metabolism; D018506:Gene Expression Regulation, Plant; D008958:Models, Molecular; D008968:Molecular Conformation; D008969:Molecular Sequence Data; D009251:NADPH-Ferrihemoprotein Reductase / Q000096:biosynthesis / Q000235:genetics*; D016366:Open Reading Frames; D010802:Phylogeny; D018515:Plant Leaves / Q000378:metabolism; D018517:Plant Roots / Q000378:metabolism; D018547:Plant Stems / Q000378:metabolism; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D014364:Tryptophan / Q000737:chemistry
chemical_list: D000970:Antineoplastic Agents; D018076:DNA, Complementary; D011994:Recombinant Proteins; D014364:Tryptophan; D045304:Cytochromes c; D009251:NADPH-Ferrihemoprotein Reductase; D002166:Camptothecin