Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant
文献类型：期刊论文
| 作者 | Qu, Xixing; Pu, Xiang; Chen, Fei; Yang, Yun; Yang, Lixia; Zhang, Guolin; Luo, Yinggang |
| 刊名 | PLOS ONE
 |
| 出版日期 | 2015 |
| 卷号 | 10期号:8页码:- |
| 关键词 | INDOLE ALKALOID BIOSYNTHESIS CATHARANTHUS-ROSEUS SYNTHASE ENZYME SITE |
| 产权排序 | 1 |
| 通讯作者 | Luo, YG (reprint author), Chinese Acad Sci, Chengdu Inst Biol, Ctr Nat Prod Res, Chengdu, Peoples R China. |
| 合作状况 | 国内 |
| 英文摘要 | 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 similar to 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- similar to 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. |
| 学科主题 | Science & Technology - Other Topics |
| 类目[WOS] | Multidisciplinary Sciences |
| 语种 | 英语 |
| 源URL | [http://210.75.237.14/handle/351003/27572]  |
| 专题 | 成都生物研究所_天然产物研究 |
| 推荐引用方式 GB/T 7714 | Qu, Xixing,Pu, Xiang,Chen, Fei,et al. Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant[J]. PLOS ONE,2015,10(8):-. |
| APA | Qu, Xixing.,Pu, Xiang.,Chen, Fei.,Yang, Yun.,Yang, Lixia.,...&Luo, Yinggang.(2015).Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant.PLOS ONE,10(8),-. |
| MLA | Qu, Xixing,et al."Molecular Cloning, Heterologous Expression, and Functional Characterization of an NADPH-Cytochrome P450 Reductase Gene from Camptotheca acuminata, a Camptothecin-Producing Plant".PLOS ONE 10.8(2015):-. |
入库方式： OAI收割
来源：成都生物研究所
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