1987 Fiscal Year Final Research Report Summary
Creation and design of novel enzymes by analysis and modification of nucleotide sequence in DNA and its application
| Project/Area Number |
61480057
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| Research Category |
Grant-in-Aid for General Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Research Field |
発酵・醸造
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| Research Institution | Kyoto University |
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Principal Investigator |
KIMURA Akira Professor:Research Institute for food Science,Kyoto University, 食糧科学研究所, 教授 (80026541)
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| Co-Investigator(Kenkyū-buntansha) |
SAKAGUCHI Morihiko Associate professor:Research Institute for food Science,Kyoto University, 食糧科学研究所, 助教授 (00027187)
ODA Jun'ichi Professor:Chemical Institute,Kyoto University, 化学研究所, 教授 (50027041)
MURATA Kousaku Research associate:Research Institute for food Science,Kyoto University, 食糧科学研究所, 助手 (90142299)
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| Project Period (FY) |
1986 – 1987
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| Keywords | <gamma>-Glutamylcysteine synthetase / Glutathione synthetase / Amino acid sequence Dihydrofolate reductase / Nucleotide sequence / DNA塩基配列 / 位置特異的点突然変異 |
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| Research Abstract |
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%.
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