Functional characterization and differential expression studies of squalene synthase from Withania somnifera.
Squalene synthase (SQS: EC 2.5.1.21) 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.
Plant Tissue Culture Division, National Chemical Laboratory, Dr. Homi Bhabha Road, Pune 411008, Maharashtra, India.
Neha
N
Gupta
Poonam
P
Sharma
R J
RJ
Santosh Kumar
Rishi K
RK
Vishwakarma
B M
BM
Khan
D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D013186:Farnesyl-Diphosphate Farnesyltransferase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism*; D018506:Gene Expression Regulation, Plant; D008969:Molecular Sequence Data; D010802:Phylogeny; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D032341:Withania / Q000145:classification / Q000235:genetics* / Q000378:metabolism*
D018076:DNA, Complementary; D011994:Recombinant Proteins; D013186:Farnesyl-Diphosphate Farnesyltransferase