Functional characterization and differential expression studies of squalene synthase from Withania somnifera
- 693 Downloads
Squalene synthase (SQS: EC 18.104.22.168) is a potential branch point regulatory enzyme and represents the first committed step to diverge the carbon flux from the main isoprenoid pathway towards sterol biosynthesis. In the present study, cloning and characterization of Withania somnifera squalene synthase (WsSQS) cDNA was investigated subsequently followed by its heterologous expression and preliminary enzyme activity. Two different types of WsSQS cDNA clones (WsSQS1and WsSQS2) were identified that contained an open reading frames of 1,236 and 1,242 bp encoding polypeptides of 412 and 414 amino acids respectively. Both WsSQS isoforms share 99 % similarity and identity with each other. WsSQS deduced amino acids sequences, when compared with SQS of other plant species, showed maximum similarity and identity with Capsicum annuum followed by Solanum tuberosum and Nicotiana tabacum. To obtain soluble recombinant enzymes, 24 hydrophobic amino acids were deleted from the carboxy terminus and expressed as 6X His–Tag fusion protein in Escherichia coli. Approximately 43 kDa recombinant protein was purified using Ni–NTA affinity chromatography and checked on SDS-PAGE. Preliminary activity of the purified enzymes was determined and the products were analyzed by gas chromatograph–mass spectrometer (GC–MS). Quantitative real-time PCR (qRT-PCR) analysis showed that WsSQS expresses more in young leaves than mature leaves, stem and root.
KeywordsWithania somnifera Squalene synthase Gas chromatograph–Mass Spectrometer (GC–MS) qRT-PCR
Squalene synthase gene
Tert-butyl methyl ether
Quantitative real-time PCR
The authors thank Dr. H. V. Thulasiram, Organic chemistry, National Chemical Laboratory (Pune, India) for providing GC–MS facility; Council of Scientific and Industrial Research (CSIR), New Delhi, India for financial support and University Grants Commission (UGC), New Delhi, India for providing fellowship.
- 3.Matsuda H, Murakami T, Kishi A, Yoshikawa M (2001) Structures of withanolides I, II, III, IV, V, VI, and VII, new withanolide glycosides, from the roots of Indian Withania somnifera DUNAL and inhibitory activity for tachyphylaxis to clonidine in isolated guinea-pig ileum. Bioorg Med Chem 9(6):1499–1507CrossRefPubMedGoogle Scholar
- 6.Bhattacharya A, Ghosal S, Bhattacharya SK (2001) Anti-oxidant effect of Withania somnifera glycowithanolides in chronic footshock stress-induced perturbations of oxidative free radical scavenging enzymes and lipid peroxidation in rat frontal cortex and striatum. J Ethnopharmacol 74(1):1–6CrossRefPubMedGoogle Scholar
- 23.Sealey-Cardona M, Cammerer S, Jones S, Ruiz-Perez LM, Brun R, Gilbert IH, Urbina JA, Gonzalez-Pacanowska D (2007) Kinetic characterization of squalene synthase from Trypanosoma cruzi: selective inhibition by quinuclidine derivatives. Antimicrob Agents Chemother 51(6):2123–2129. doi: 10.1128/AAC.01454-06 CrossRefPubMedPubMedCentralGoogle Scholar
- 29.Uchida H, Yamashita H, Kajikawa M, Ohyama K, Nakayachi O, Sugiyama R, Yamato KT, Muranaka T, Fukuzawa H, Takemura M (2009) Cloning and characterization of a squalene synthase gene from a petroleum plant, Euphorbia tirucalli L. Planta 229(6):1243–1252. doi: 10.1007/s00425-009-0906-6 CrossRefPubMedGoogle Scholar
- 30.Kribii R, Arro M, Del Arco A, Gonzalez V, Balcells L, Delourme D, Ferrer A, Karst F, Boronat A (1997) Cloning and characterization of the Arabidopsis thaliana SQS1 gene encoding squalene synthase—involvement of the C-terminal region of the enzyme in the channeling of squalene through the sterol pathway. Eur J Biochem 249(1):61–69CrossRefPubMedGoogle Scholar
- 42.Freeman WM, Walker SJ, Vrana KE (1999) Quantitative RT-PCR: pitfalls and potential. Biotechniques 26(1):112–122, 124–115Google Scholar
- 45.Pandit J, Danley DE, Schulte GK, Mazzalupo S, Pauly TA, Hayward CM, Hamanaka ES, Thompson JF, Harwood HJ, Jr (2000) Crystal structure of human squalene synthase. A key enzyme in cholesterol biosynthesis. J Biol Chem 275(39):30610–30617. doi: 10.1074/jbc.M004132200M004132200 CrossRefPubMedGoogle Scholar