Related Experiment Videos
Purification, characterization, and cDNA cloning of an NADPH-cytochrome P450 reductase from mung bean
M S Shet1, K Sathasivan, M A Arlotto
1Department of Biochemistry, University of Texas Southwestern Medical Center, Dallas 75235.
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.
Insights
Mung bean seedlings yielded NADPH-cytochrome P450 reductase, a flavoprotein crucial for P450 activity. This plant enzyme shares functional similarities with mammalian counterparts, despite significant sequence divergence.
Area of Science:
- Biochemistry
- Plant Molecular Biology
- Enzymology
Background:
- Microsomal fractions of etiolated mung bean seedlings (Vigna radiata var. Berken) contain NADPH-cytochrome P450 reductase (EC 1.6.2.4).
- This flavoprotein plays a vital role in various metabolic pathways, including drug metabolism and steroidogenesis, through its interaction with cytochrome P450 enzymes.
Purpose of the Study:
- To isolate and characterize the NADPH-cytochrome P450 reductase from mung bean seedlings.
- To determine its amino acid sequence and compare it with mammalian reductases.
- To investigate its functional activity in reconstituted P450 systems.
Main Methods:
- 1150-fold purification of the plant reductase using biochemical techniques.
- SDS/PAGE for molecular mass determination.
- Western blot analysis and antibody inhibition studies for epitope comparison.
- Peptide sequencing and PCR amplification for cDNA cloning and sequence analysis.
Main Results:
- A predominant protein band of approximately 82 kDa was identified.
- The purified enzyme is a glycoprotein with characteristic flavoprotein absorbance spectrum, containing equimolar FAD and FMN.
- Unique epitopes distinguish plant from mammalian reductases, yet the mung bean enzyme functionally substituted for rat reductase in a P450-catalyzed reaction.
Conclusions:
- The mung bean NADPH-cytochrome P450 reductase shares conserved functional domains with mammalian enzymes despite only 38% sequence identity.
- This plant reductase can functionally replace its mammalian counterpart in specific P450-mediated reactions, highlighting potential cross-species functional conservation.