Functional characterization of two distinct classes of NADPH-cytochrome P450 reductases in Senna alexandrina Mill.

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Senna alexandrina Mill., an important medicinal plant of Fabaceae family, is famous for its laxative properties which are mainly due to the presence of sennosides (anthraquinone glycosides). However, the complete biosynthetic pathway of sennosides in Senna is not yet fully understood. Cytochrome P450 monooxygenases (CYPs), which are heme-containing enzymes are supposed to play key roles in sennoside biosynthesis. Cytochrome P450 reductases (CPRs) are essential for the activity of CYPs, as they function as their redox partners. However, CPRs in Senna have not yet been characterized in detail. In this study, two different sequences of SaCPRs were retrieved from the publicly available Transcriptome Shotgun Assembly (TSA) database of S. alexandrina at National Center for Biotechnology Information (NCBI). The open reading frames of SaCPR1 and SaCPR2 were found to be 2079 and 2121bp, encoding 693 and 707 amino acid long polypeptides, respectively. Phylogenetic and 3-D structure analysis predicted that these two SaCPRs (i.e. SaCPR1 and SaCPR2) were grouped with the members of Class I and Class II CPRs, respectively. Analysis of SaCPR1 and SaCPR2 sequences showed that the conserved domains commonly found in CPRs such as FMN- (Flavin adenine mononucleotide), FAD-(Flavin adenine dinucleotide), NADPH-(Nicotinamide adenine dinucleotide phosphate hydrogen) and cytochrome P450 binding region, were also present in SaCPRs. SaCPR1 and SaCPR2 were cloned and expressed in yeast for functional characterization. In cytochrome P450 reductase assay, both SaCPR1 and SaCPR2 reduced cytochrome c in the presence of NADPH as an electron donor, however, SaCPR1 showed higher specific activity than SaCPR2. The real time expression analysis of SaCPRs performed in the leaf, stem and root tissues of Senna showed that SaCPR1 was expressed more in leaf tissue while SaCPR2 expressed more in stem tissue. Furthermore, both the SaCPRs were found to be induced by salicylic acid as well as wound treatment (up to two hr). Two different classes of cytochrome P450 reductases were identified and functionally characterized. SaCPR1 showed higher in vitro activity than SaCPR2 in cytochrome c reduction assay.