Molecular Biology Reports

, Volume 39, Issue 9, pp 8803–8812 | Cite as

Functional characterization and differential expression studies of squalene synthase from Withania somnifera

  • Neha Gupta
  • Poonam Sharma
  • R. J. Santosh Kumar
  • Rishi K. Vishwakarma
  • B. M. Khan


Squalene synthase (SQS: EC 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.


Withania somnifera Squalene synthase Gas chromatograph–Mass Spectrometer (GC–MS) qRT-PCR 



Isopropyl β-d-1-thiogalactopyranoside


Squalene synthase


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.


  1. 1.
    Singh B, Saxena AK, Chandan BK, Gupta DK, Bhutani KK, Anand KK (2001) Adaptogenic activity of a novel, withanolide-free aqueous fraction from the roots of Withania somnifera Dun. Phytother Res 15(4):311–318. doi: 10.1002/ptr.858 CrossRefPubMedGoogle Scholar
  2. 2.
    Misra L, Lal P, Sangwan RS, Sangwan NS, Uniyal GC, Tuli R (2005) Unusually sulfated and oxygenated steroids from Withania somnifera. Phytochemistry 66(23):2702–2707. doi: 10.1016/j.phytochem.2005.10.001 CrossRefPubMedGoogle Scholar
  3. 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
  4. 4.
    Ray AB, Gupta M (1994) Withasteroids, a growing group of naturally occurring steroidal lactones. Fortschr Chem Org Naturst 63:1–106PubMedGoogle Scholar
  5. 5.
    Gupta SK, Dua A, Vohra BP (2003) Withania somnifera (Ashwagandha) attenuates antioxidant defense in aged spinal cord and inhibits copper induced lipid peroxidation and protein oxidative modifications. Drug Metabol Drug Interact 19(3):211–222CrossRefPubMedGoogle Scholar
  6. 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
  7. 7.
    Kulkarni RR, Patki PS, Jog VP, Gandage SG, Patwardhan B (1991) Treatment of osteoarthritis with a herbomineral formulation: a double-blind, placebo-controlled, cross-over study. J Ethnopharmacol 33(1–2):91–95CrossRefPubMedGoogle Scholar
  8. 8.
    Devi PU, Sharada AC, Solomon FE, Kamath MS (1992) In vivo growth inhibitory effect of Withania somnifera (Ashwagandha) on a transplantable mouse tumor, Sarcoma 180. Indian J Exp Biol 30(3):169–172PubMedGoogle Scholar
  9. 9.
    Sharada AC, Solomon FE, Devi PU, Udupa N, Srinivasan KK (1996) Antitumor and radiosensitizing effects of withaferin A on mouse Ehrlich ascites carcinoma in vivo. Acta Oncol 35(1):95–100CrossRefPubMedGoogle Scholar
  10. 10.
    Dhuley JN (1997) Effect of some Indian herbs on macrophage functions in ochratoxin A treated mice. J Ethnopharmacol 58(1):15–20CrossRefPubMedGoogle Scholar
  11. 11.
    Archana R, Namasivayam A (1999) Antistressor effect of Withania somnifera. J Ethnopharmacol 64(1):91–93CrossRefPubMedGoogle Scholar
  12. 12.
    Davis L, Kuttan G (1998) Suppressive effect of cyclophosphamide-induced toxicity by Withania somnifera extract in mice. J Ethnopharmacol 62(3):209–214CrossRefPubMedGoogle Scholar
  13. 13.
    Mishra L-C, Singh BB, Dagenais S (2000) Scientific basis for the therapeutic use of Withania somnifera (Ashwagandha): a review. Altern Med Rev 5(4):334–346PubMedGoogle Scholar
  14. 14.
    Atal CK, Dhar KL, Singh J (1975) The chemistry of Indian Piper species. Lloydia 38(3):256–264PubMedGoogle Scholar
  15. 15.
    Paradise EM, Kirby J, Chan R, Keasling JD (2008) Redirection of flux through the FPP branch-point in Saccharomyces cerevisiae by down-regulating squalene synthase. Biotechnol Bioeng 100(2):371–378. doi: 10.1002/bit.21766 CrossRefPubMedGoogle Scholar
  16. 16.
    Shimada H, Kondo K, Fraser PD, Miura Y, Saito T, Misawa N (1998) Increased carotenoid production by the food yeast Candida utilis through metabolic engineering of the isoprenoid pathway. Appl Environ Microbiol 64(7):2676–2680PubMedPubMedCentralGoogle Scholar
  17. 17.
    Lee S, Poulter CD (2008) Cloning, solubilization, and characterization of squalene synthase from Thermosynechococcus elongatus BP-1. J Bacteriol 190(11):3808–3816. doi: 10.1128/JB.01939-07 CrossRefPubMedPubMedCentralGoogle Scholar
  18. 18.
    Jennings SM, Tsay YH, Fisch TM, Robinson GW (1991) Molecular cloning and characterization of the yeast gene for squalene synthetase. Proc Natl Acad Sci USA 88(14):6038–6042CrossRefPubMedPubMedCentralGoogle Scholar
  19. 19.
    LoGrasso PV, Soltis DA, Boettcher BR (1993) Overexpression, purification, and kinetic characterization of a carboxyl-terminal-truncated yeast squalene synthetase. Arch Biochem Biophys 307(1):193–199. doi: 10.1006/abbi.1993.1578 CrossRefPubMedGoogle Scholar
  20. 20.
    Zhao MW, Liang WQ, Zhang DB, Wang N, Wang CG, Pan YJ (2007) Cloning and characterization of squalene synthase (SQS) gene from Ganoderma lucidum. J Microbiol Biotechnol 17(7):1106–1112PubMedGoogle Scholar
  21. 21.
    Inoue T, Osumi T, Hata S (1995) Molecular cloning and functional expression of a cDNA for mouse squalene synthase. Biochim Biophys Acta 1260(1):49–54CrossRefPubMedGoogle Scholar
  22. 22.
    McKenzie TL, Jiang G, Straubhaar JR, Conrad DG, Shechter I (1992) Molecular cloning, expression, and characterization of the cDNA for the rat hepatic squalene synthase. J Biol Chem 267(30):21368–21374PubMedGoogle Scholar
  23. 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
  24. 24.
    Robinson GW, Tsay YH, Kienzle BK, Smith-Monroy CA, Bishop RW (1993) Conservation between human and fungal squalene synthetases: similarities in structure, function, and regulation. Mol Cell Biol 13(5):2706–2717CrossRefPubMedPubMedCentralGoogle Scholar
  25. 25.
    Summers C, Karst F, Charles AD (1993) Cloning, expression and characterisation of the cDNA encoding human hepatic squalene synthase, and its relationship to phytoene synthase. Gene 136(1-2Che):185–192CrossRefPubMedGoogle Scholar
  26. 26.
    Devarenne TP, Ghosh A, Chappell J (2002) Regulation of squalene synthase, a key enzyme of sterol biosynthesis, in tobacco. Plant Physiol 129(3):1095–1106. doi: 10.1104/pp.001438 CrossRefPubMedPubMedCentralGoogle Scholar
  27. 27.
    Lee MH, Jeong JH, Seo JW, Shin CG, Kim YS, In JG, Yang DC, Yi JS, Choi YE (2004) Enhanced triterpene and phytosterol biosynthesis in Panax ginseng overexpressing squalene synthase gene. Plant Cell Physiol 45(8):976–984. doi: 10.1093/pcp/pch12645/8/976 CrossRefPubMedGoogle Scholar
  28. 28.
    Hayashi H, Hirota A, Hiraoka N, Ikeshiro Y (1999) Molecular cloning and characterization of two cDNAs for Glycyrrhiza glabra squalene synthase. Biol Pharm Bull 22(9):947–950CrossRefPubMedGoogle Scholar
  29. 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. 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
  31. 31.
    Yoshioka H, Yamada N, Doke N (1999) cDNA cloning of sesquiterpene cyclase and squalene synthase, and expression of the genes in potato tuber infected with Phytophthora infestans. Plant Cell Physiol 40(9):993–998CrossRefPubMedGoogle Scholar
  32. 32.
    Zhang D, Jennings SM, Robinson GW, Poulter CD (1993) Yeast squalene synthase: expression, purification, and characterization of soluble recombinant enzyme. Arch Biochem Biophys 304(1):133–143CrossRefPubMedGoogle Scholar
  33. 33.
    Jiang G, McKenzie TL, Conrad DG, Shechter I (1993) Transcriptional regulation by lovastatin and 25-hydroxycholesterol in HepG2 cells and molecular cloning and expression of the cDNA for the human hepatic squalene synthase. J Biol Chem 268(17):12818–12824PubMedGoogle Scholar
  34. 34.
    Sasiak K, Rilling HC (1988) Purification to homogeneity and some properties of squalene synthetase. Arch Biochem Biophys 260(2):622–627CrossRefPubMedGoogle Scholar
  35. 35.
    Belingheri L, Beyer P, Kleinig H, Gleizes M (1991) Solubilization and partial purification of squalene synthase from daffodil microsomal membranes. FEBS Lett 292(1–2):34–36PubMedGoogle Scholar
  36. 36.
    Hanley K, Chappell J (1992) Solubilization, partial purification, and immunodetection of squalene synthetase from tobacco cell suspension cultures. Plant Physiol 98(1):215–220CrossRefPubMedPubMedCentralGoogle Scholar
  37. 37.
    Lee JH, Yoon YH, Kim HY, Shin DH, Kim DU, Lee IJ, Kim KU (2002) Cloning and expression of squalene synthase cDNA from hot pepper (Capsicum annuum L.). Mol Cells 13(3):436–443PubMedGoogle Scholar
  38. 38.
    Tamura K, Dudley J, Nei M, Kumar S (2007) MEGA4: molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. Mol Biol Evol 24(8):1596–1599. doi: 10.1093/molbev/msm092 CrossRefPubMedGoogle Scholar
  39. 39.
    Saitou N, Nei M (1987) The neighbor-joining method: a new method for reconstructing phylogenetic trees. Mol Biol Evol 4(4):406–425PubMedGoogle Scholar
  40. 40.
    Kim O-T, Seong N-S, Kim M-Y, Hwang B (2005) Isolation and characterization of squalene synthase cDNA from Centella asiatica (L.). Urban J Plant Biol 48(3):263–269CrossRefGoogle Scholar
  41. 41.
    Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254CrossRefPubMedGoogle Scholar
  42. 42.
    Freeman WM, Walker SJ, Vrana KE (1999) Quantitative RT-PCR: pitfalls and potential. Biotechniques 26(1):112–122, 124–115Google Scholar
  43. 43.
    Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29(9):e45CrossRefPubMedPubMedCentralGoogle Scholar
  44. 44.
    Chen A, Kroon PA, Poulter CD (1994) Isoprenyl diphosphate synthases: protein sequence comparisons, a phylogenetic tree, and predictions of secondary structure. Protein Sci 3(4):600–607. doi: 10.1002/pro.5560030408 CrossRefPubMedPubMedCentralGoogle Scholar
  45. 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
  46. 46.
    Reipen IG, Poralla K, Sahm H, Sprenger GA (1995) Zymomonas mobilis squalene-hopene cyclase gene (shc): cloning, DNA sequence analysis, and expression in Escherichia coli. Microbiology 141(Pt 1):155–161CrossRefPubMedGoogle Scholar
  47. 47.
    Marrero PF, Poulter CD, Edwards PA (1992) Effects of site-directed mutagenesis of the highly conserved aspartate residues in domain II of farnesyl diphosphate synthase activity. J Biol Chem 267(30):21873–21878PubMedGoogle Scholar
  48. 48.
    Huang Z, Jiang K, Pi Y, Hou R, Liao Z, Cao Y, Han X, Wang Q, Sun X, Tang K (2007) Molecular cloning and characterization of the yew gene encoding squalene synthase from Taxus cuspidata. J Biochem Mol Biol 40(5):625–635CrossRefPubMedGoogle Scholar
  49. 49.
    Yin T, Cao X, Miao Q, Li C, Chen X, Zhao M, Jiang J (2011) Molecular cloning and functional analysis of an organ-specific expressing gene coding for farnesyl diphosphate synthase from Michelia chapensis Dandy. Acta Physiol Plant 33:137–144. doi: 10.1007/s11738-010-0529-3 CrossRefGoogle Scholar
  50. 50.
    Gupta P, Akhtar N, Tewari SK, Sangwan RS, Trivedi PK (2011) Differential expression of farnesyl diphosphate synthase gene from Withania somnifera in different chemotypes and in response to elicitors. Plant Growth Regul 65:93–100. doi: 10.1007/s10725-011-9578-x CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media B.V. 2012

Authors and Affiliations

  • Neha Gupta
    • 1
  • Poonam Sharma
    • 1
  • R. J. Santosh Kumar
    • 1
  • Rishi K. Vishwakarma
    • 1
  • B. M. Khan
    • 1
  1. 1.Plant Tissue Culture DivisionNational Chemical LaboratoryPuneIndia

Personalised recommendations