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Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean.

ABSTRACT: We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhibited a typical flavoprotein visible absorbance spectrum, and contained almost equimolar quantities of FAD and FMN per mole of enzyme. Specific antibodies revealed the presence of unique epitopes distinguishing the plant and mammalian flavoproteins as demonstrated by Western blot analyses and inhibition studies. Peptide fragments from the purified plant NADPH-P450 reductase were sequenced, and degenerate primers were used in PCR amplification reactions. Overlapping cDNA clones were sequenced, and the deduced amino acid sequence of the mung bean NADPH-P450 reductase was compared with equivalent enzymes from mammalian species. Although common flavin and NADPH-binding sites are recognizable, there is only approximately 38% amino acid sequence identity. Surprisingly, the purified mung bean NADPH-P450 reductase can substitute for purified rat NADPH-P450 reductase in the reconstitution of the mammalian P450-catalyzed 17 alpha-hydroxylation of pregnenolone or progesterone.

SUBMITTER: Shet MS

PROVIDER: S-EPMC46202 | biostudies-other | 1993 Apr

REPOSITORIES: biostudies-other

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Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean.

Shet M S MS Sathasivan K K Arlotto M A MA Mehdy M C MC Estabrook R W RW

Proceedings of the National Academy of Sciences of the United States of America 19930401 7

We report here the isolation and deduced amino acid sequence of the flavoprotein, NADPH-cytochrome P450 (cytochrome c) reductase (EC 1.6.2.4), associated with the microsomal fraction of etiolated mung bean seedlings (Vigna radiata var. Berken). An 1150-fold purification of the plant reductase was achieved, and SDS/PAGE showed a predominant protein band with an apparent molecular mass of approximately 82 kDa. The purified plant NADPH-P450 reductase gave a positive reaction as a glycoprotein, exhi ...[more]

PMID: 8464904

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Project description: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.

Dataset Information

Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean.

Publications

Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean.

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