| Project/Area Number |
03680172
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| Research Category |
Grant-in-Aid for General Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Research Field |
代謝生物化学
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| Research Institution | Kochi University |
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Principal Investigator |
MISONO Haruo Kochi University,Agriculture,Professor, 農学部, 教授 (30027073)
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| Co-Investigator(Kenkyū-buntansha) |
NAGATA Shinji Kochi University,Agriculture,Assistant Prof., 農学部, 助教授 (60180494)
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| Project Period (FY) |
1991 – 1992
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| Project Status |
Completed (Fiscal Year 1992)
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| Budget Amount *help |
¥1,900,000 (Direct Cost: ¥1,900,000)
Fiscal Year 1992: ¥900,000 (Direct Cost: ¥900,000)
Fiscal Year 1991: ¥1,000,000 (Direct Cost: ¥1,000,000)
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| Keywords | Lysine dehydrogenase / Chemical modification / Cloning / DNA sequence / Amino acid sequence / Primary structure / クロ-ニング |
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| Research Abstract |
Lysine dehydrogenase is a unique enzyme which is activated by the substrate L-lysine. L-Lysine induced association of the dimeric enzymes into tetramers. The enzyme has effector-binding sites in addition to the catalytic site. In order to know amino acid residues in the catalytic site and the effector-binding site, chemical modification of the dimeric and tetrameric enzymes were done using various reagents. The results suggest that arginine and tryptophan residues are present in the catalytic site and lysine and cysteine residues are present in the effector-binding site.
Agrobacterium tumefaciens has Ti-plasmids, but the structural gene of this enzyme was not coded by Ti-plasmid. The chromosomal DNA of the bacterium was digested partially with a restriction endonuclease, HindIII and cloned into E. coli JM109 using pUC18 as a vector. One cloned cell which showed the enzyme activity was selected from 4,000 cloned cells. The plasmid obtained from this cell, pKUKD19, has a inserted DNA fragment (about 3.5kb). The pKUKD19-4 which has a SspI-XhoI fragment (about 1.3kb) was derived by subcloning. This fragment was inserted into a high expression vector, pKK223-3 and the E. coli cell which produces the enzyme aboundantly was obtained. The DNA fragment was sequenced and the primary structure of the enzyme was determined. The NAD binding domain was located in the N-terminal region and the catalytic domain was in the C-terminal region. The lysine residue (G-G-G-K) which functions in catalysis in other amino acid dehydrogenases was not found in this enzyme.
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