Creation and design of novel enzymes by analysis and modification of nucleotide sequence in DNA and its application

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Glutathione synthetase (GSH-II) of Eschrichia coli B catalyzes the synthesis of glutathione from <gamma>-glutamylcysteine and glycine in the Presence of adenosine 5'-triphosphate (ATP).To analyze the relation between function and structure of the enzyme,we first searched for proteins with a homologous amino acid sequence with that of GSH-II and found that an enzyme,dihydroxyfolate reductase(FDR)has a region approximately 70% homologous in amino acid sequence.The enzymatic analyses using inhibitors and/or substrates of the two enzymes (GSH-II and FDR) indicated that the homologous region represented the catalytic domain on the two enzymes.Secondly,to know the contribution of cysteine residues on activity and/or stability of the GSH-II,the 4 codons for cysteine in the GSH-II subunit were severally changed to the codons for alanine by site-directed mutagenesis.Although either replacement did not affect the activity,the replacements of more than 2 cysteine residues resulted in the significant decrease in the GSH-II activity. The 4 cysteine residues in the GSH-II subunit were,therefore,thought to play an important role in the full expression of the GSH-II activity.<gamma>-Glutamylcysteine synthetase(GSH-I)catalyzes the first step of glutathione synthesis and produces <gamma>-glutamylcysteine from glutamate and cysteine in the presence of ATP.The gene for the GSH-I has an unusual initiation codon TTG.The codon (TTG)was replaced with general initition codon ATG by site-directed mutagenesis.As a result, the transcriptional efficiency of the gene was increased more than 50%.