Site-Specific Mutagenesis of the gshI Gene for Increasing the Activity of ${\gamma}$ -Glutamylcysteine Synthetase in Escherichia coli K-12

The gshI gene from the Escherichia coli K-12 strain codes for synthetase which mediates the rate-limiting step of glutathione biosynthesis. The isolated gshI gene from E. coli K-12 has an unusual translation initiation codon, UUG. The 494th amino acid is Ala rather than Gly which was found in a mutant strain E. coli B. In order to improve the translational rate of the gshI gene of E. coli K-12, the initiation codon, UUG, was changed to the usual AUG codon by the site-specific mutagenesis. This change has resulted in a 53% increase of synthetase activity. The enzyme activity was also improved by replacing with Val (A494V) or Leu (A494L). The replacement of with Thr (S495T) also resulted in a 62% increase of the enzyme activity. Therefore, the specific activity of synthetase was increased with the increasing chain length of the aliphathic amino acid at the site of the 494th amino acid (Ala).