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Characterization of Plant S-Adenosyl-L-Methionine Decarboxylase and Spermidine Synthase in Polyamine Deficient Mutant Strain of E. Coli

Author Affiliations

  • 1Depatment of Botany, Bethune College, 181 Bidhan Sarani, Kolkata-700006, INDIA
  • 2Molecular & Human Genetics Division, Indian Institute of Chemical Biology 4, Raja S.C.Mullick Road, Kolkata 700032, INDIA
  • 3Division of Plant Biology, Bose Institute, 93/1 A.P.C Road, Kolkata- 700 009, INDIA

Int. Res. J. Biological Sci., Volume 3, Issue (8), Pages 60-68, August,10 (2014)

Abstract

S-adenosylmethionine decarboxylase (SAMdC, EC 4.1.1.50) and Spermidine synthase (SPDS, EC 2.5.1.1), two enzymes of plant polyamine biosynthetic pathway were cloned to gain further insight on the polyamine metabolism through a molecular approach. cDNAs for SAMdC and SPDS were isolated from Oryza sativa cv. Pokkali and Nicotiana tabacum cv. Jayasri respectively. Rice SAMdC (rSAMdC) and Tobacco SPDS (tSPDS) proteins were overexpressed in E. coli using expression vectors pEZZ18 and pGEX-3X respectively. N-terminally protein-A-tagged 43.8 kDa pre-protein of rSAMdC and Nterminally GST-tagged 34.7 kDa of tSPDS were purified by affinity chromatography. The activities of the recombinant proteins were confirmed by the appearance of spermidine, product of the coupled reaction involving rSAMdC and tSPDS. As a direct evidence of the function of these plant enzymes, the complementation assay using extract of E. coli mutant strain, HT252 (F-?(speAspeB) ?(speCglc) ?(speED) thr-1 proA2 thi-1 lacY1 galK2 m-), deficient in polyamine biosynthetic enzymes was performed. Reconstitution of the E. coli polyamine biosynthetic pathway by two different plant enzymes rSAMdC and tSPDS, simultaneously supplemented with exogenous S-adenosylmethionine and putrescine, was the novel observation of the present work.

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