title: Light and auxin responsive cytochrome P450s from Withania somnifera Dunal: cloning, expression and molecular modelling of two pairs of homologue genes with differential regulation.
abstract: Cytochrome P450s (CYPs) catalyse a wide variety of oxygenation/hydroxylation reactions that facilitate diverse metabolic functions in plants. Specific CYP families are essential for the biosynthesis of species-specialized metabolites. Therefore, we investigated the role of different CYPs related to secondary metabolism in Withania somnifera, a medicinally important plant of the Indian subcontinent. In this study, complete complementary DNAs (cDNAs) of four different CYP genes were isolated and christened as WSCYP93Id, WSCYP93Sm, WSCYP734B and WSCYP734R. These cDNAs encoded polypeptides comprising of 498, 496, 522 and 550 amino acid residues with their deduced molecular mass of 56.7, 56.9, 59.4 and 62.2 kDa, respectively. Phylogenetic study and molecular modelling analysis of the four cloned WSCYPs revealed their categorization into two CYP families (CYP83B1 and CYP734A1) belonging to CYP71 and CYP72 clans, respectively. BLASTp searches showed similarity of 75 and 56 %, respectively, between the two CYP members of CYP83B1 and CYP734A1 with major variances exhibited in their N-terminal regions. The two pairs of homologues exhibited differential expression profiles in the leaf tissues of selected chemotypes of W. somnifera as well as in response to treatments such as methyl jasmonate, wounding, light and auxin. Light and auxin regulated two pairs of WSCYP homologues in a developing seedling in an interesting differential manner. Their lesser resemblance and homology with other CYP sequences suggested these genes to be more specialized and distinct ones. The results on chemotype-specific expression patterns of the four genes strongly suggested their key/specialized involvement of the CYPs in the biosynthesis of chemotype-specific metabolites, though their further biochemical characterization would reveal the specificity in more detail. It is revealed that WSCYP93Id and WSCYP93Sm may be broadly involved in the oxygenation reactions in the plant and, thereby, control various pathways involving such metabolic reactions in the plant. As a representative experimental validation of this notion, WSCYP93Id was heterologouly expressed in Escherichia coli and catalytic capabilities of the recombinant WSCYP93Id protein were evaluated using withanolides as substrates. Optimized assays with some major withanolides (withanone, withaferin A and withanolide A) involving spectrophotometric as well as high-pressure liquid chromatography (HPLC)-based evaluation (product detection) of the reactions showed conversion of withaferin A to a hydroxylated product. The genes belonging to other CYP group are possibly involved in some specialised synthesis such as that of brassinosteroids.
authors: Metabolic and Structural Biology Department, CSIR-Central Institute for Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, UP, India.|Jacob Blaustein Institute for Desert Research, Albert Katz Department of Dryland Biotechnologies, Ben-Gurion University of the Negev, Negev, Israel.
authors: Sudhakar
authors: S
authors: Srivastava
authors: Metabolic and Structural Biology Department, CSIR-Central Institute for Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, UP, India. sangwan.lab@gmail.com.|Centre of Innovative and Applied Bioprocessing (CIAB), (A National Institute under Department of Biotechnology, Government of India), Mohali, 1600 71, Punjab, India. sangwan.lab@gmail.com.
authors: Rajender Singh
authors: RS
authors: Sangwan
authors: Metabolic and Structural Biology Department, CSIR-Central Institute for Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, UP, India.
authors: Sandhya
authors: S
authors: Tripathi
authors: Metabolic and Structural Biology Department, CSIR-Central Institute for Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, UP, India.
authors: Bhawana
authors: B
authors: Mishra
authors: Metabolic and Structural Biology Department, CSIR-Central Institute for Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, UP, India.
authors: L K
authors: LK
authors: Narnoliya
authors: Metabolic and Structural Biology Department, CSIR-Central Institute for Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, UP, India.
authors: L N
authors: LN
authors: Misra
authors: Metabolic and Structural Biology Department, CSIR-Central Institute for Medicinal and Aromatic Plants (CSIR-CIMAP), Lucknow, 226015, UP, India. nss.cimap@gmail.com.|Centre of Innovative and Applied Bioprocessing (CIAB), (A National Institute under Department of Biotechnology, Government of India), Mohali, 1600 71, Punjab, India. nss.cimap@gmail.com.
authors: Neelam S
authors: NS
authors: Sangwan
mesh_terms: D001711:Biotransformation; D003001:Cloning, Molecular; D019295:Computational Biology; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D030541:Databases, Genetic; D015971:Gene Expression Regulation, Enzymologic; D018506:Gene Expression Regulation, Plant; D006900:Hydroxylation; D007210:Indoleacetic Acids / Q000494:pharmacology*; D007527:Isoenzymes; D008027:Light; D008958:Models, Molecular; D010802:Phylogeny; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism*; D010946:Plants, Medicinal; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000378:metabolism; D017422:Sequence Analysis, DNA; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid; D012689:Sequence Homology, Nucleic Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity; D032341:Withania / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics / Q000528:radiation effects; D054358:Withanolides / Q000378:metabolism
chemical_list: D007210:Indoleacetic Acids; D007527:Isoenzymes; D010940:Plant Proteins; D011994:Recombinant Proteins; D054358:Withanolides; D003577:Cytochrome P-450 Enzyme System
keywords: Ashwagandha; Cytochrome P450; Protein modelling; Withania somnifera; Withanolides