Abstract
A putative S-adenosylmethionine synthetase (SAM-s) gene from Streptomyces avermitilis NRRL8165 was cloned and characterized. The gene encodes a protein of 402 amino acids with a molecular weight of about 43.5 kDa. Amino acid sequence analysis revealed high sequence identity (84–93%) to known SAM-s proteins from Streptomyces species containing the conserved sequence motifs of SAM-s proteins, such as nanopeptide 276GGGAFSGKD 284 for the P-loop for ATP binding site and metal binding site ( 17D and 289D for Mg 2+ and 49G for K +). The purified protein from the transformed Escherichia coli synthesized S-adenosylmethionine (SAM) from ATP and l-methionine in vitro with a K m of 110(±32) and 340(±18) μM and V max of 7.9(±0.2) and 21.6(±1.5) μmol/mg/min for l-methionine and ATP, respectively. This result demonstrates that the isolated gene indeed encoded the SAM-s protein. The enzymatic activity of SAM-s protein was optimal at pH 8.0 and 35 °C and required divalent metal ions (Mg 2+ or Mn 2+) as a cofactor, like SAM-s proteins from other species. When the SAM-s was overexpressed in S. avermitilis, a wild type producer of avermectin, and Streptomyces peucetius, a wild type producer of doxorubicin, the production of antibiotics significantly increased by 2-fold for avermectin and 3.5-fold for doxorubicin, respectively, compared with the respective wild type strain.
Published Version
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