Cloning and Functional Characterization of NADPH-Cytochrome P450 Reductases in Aconitum vilmorinianum
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Published:2023-11-03
Issue:21
Volume:28
Page:7409
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ISSN:1420-3049
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Container-title:Molecules
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language:en
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Short-container-title:Molecules
Author:
Cheng Jingping1,
Li Guodong1,
Wang Xue1,
Yang Congwei1,
Xu Furong1ORCID,
Qian Zigang1,
Ma Xiaohui12ORCID
Affiliation:
1. College of Chinese Materia Medica, Yunnan Key Laboratory of Southern Medicinal Utilization, Yunnan University of Chinese Medicine, Kunming 650500, China
2. Key Laboratory of Yunnan Provincial Department of Education on Substance Benchmark Research of Ethnic Medicines, Kunming 650500, China
Abstract
Diterpenoid alkaloids (DAs) are major pharmacologically active ingredients of Aconitum vilmorinianum, an important medicinal plant. Cytochrome P450 monooxygenases (P450s) are involved in the DA biosynthetic pathway, and the electron transfer reaction of NADPH-cytochrome P450 reductase (CPR) with P450 is the rate-limiting step of the P450 redox reaction. Here, we identified and characterized two homologs of CPR from Aconitum vilmorinianum. The open reading frames of AvCPR1 and AvCPR2 were found to be 2103 and 2100 bp, encoding 700 and 699 amino acid residues, respectively. Phylogenetic analysis characterized both AvCPR1 and AvCPR2 as class II CPRs. Cytochrome c and ferricyanide could be reduced with the recombinant proteins of AvCPR1 and AvCPR2. Both AvCPR1 and AvCPR2 were expressed in the roots, stems, leaves, and flowers of A. vilmorinianum. The expression levels of AvCPR1 and AvCPR2 were significantly increased in response to methyl jasmonate (MeJA) treatment. The yeasts co-expressing AvCPR1/AvCPR2/SmCPR1 and CYP76AH1 all produced ferruginol, indicating that AvCPR1 and AvCPR2 can transfer electrons to CYP76AH1 in the same manner as SmCPR1. Docking analysis confirmed the experimentally deduced functional activities of AvCPR1 and AvCPR2 for FMN, FAD, and NADPH. The functional characterization of AvCPRs will be helpful in disclosing molecular mechanisms relating to the biosynthesis of diterpene alkaloids in A. vilmorinianum.
Funder
Yunnan Provincial Science and Technology Department-Applied Basic Research Joint Special Funds of Chinese Medicine
Key Project at Central Government Level: The Ability Establishment of Sustainable Use for Valuable Chinese Medicine Resources
Yunnan Science and Technology Talents and Platform Program
Wang Yuan Chao Expert Workstation in Yunnan Province
Subject
Chemistry (miscellaneous),Analytical Chemistry,Organic Chemistry,Physical and Theoretical Chemistry,Molecular Medicine,Drug Discovery,Pharmaceutical Science
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