1998 Fiscal Year Final Research Report Summary
Mechanisms of Catalytic Function of Prenyl Chain Elongating Enzymes
| Project/Area Number |
09480138
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| Research Category |
Grant-in-Aid for Scientific Research (B)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Bioorganic chemistry
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| Research Institution | Tohoku University |
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Principal Investigator |
KOYAMA Tanetoshi Tohoku University, Institute for Chemical Reaction Science, Professor, 反応化学研究所, 教授 (20089808)
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| Project Period (FY) |
1997 – 1998
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| Keywords | prenyltransferase / prenyl chain elongation / isoprenoid biosynthesis / farnesyl diphosphate / polyprenol / undecaprenyl diphosphate / heterosubunit / gene cloning |
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| Research Abstract |
1) Molecular Cloning of Undecaprenyl Diphosphate Synthase Gene and Identification of Conserved Sequences for Z-Prenyl Chain Elongating Enzymes
Cloning of the gene for undecaprenyl diphosphate synthase (UPS) was carried out by developing a novel screening method to detect colonies overexpressing extra prenyltransferases in Escherichia coli cells by autoradiography, providing the first primary structure for any prenyltransferases that catalyze Z-prenyl chain elongation. The deduced primary structure of the UPS is totally different from those of E-prenyl chain elongating enzymes which have characteristic conserved regions including aspartate-rich motifs. Construction of the gene expression system which enabled overproduction of the UPS in E.coil cells was successful. Purification and crystallization of the UPS was carried out.
2) Elucidation of the Function-Expressing Mechanisms of Heptaprenyl Diphosphate Synthase
Heptaprenyl diphosphate synthase (HepPS) of Bacillus subtilis is composed of t
… More
wo dissociable subunits, componentsand I -II, both of which are required for the catalytic function. The dynamic interaction in forming a catalytically active complex was investigated by gel filtration and immunoblotting analysis. The mechanism of this heteromeric enzyme is understood by assuming that association and dissociation of the two subunits facilitate turnover of catalysis for the synthesis of the amphipathic water-insoluble product from soluble substrates. Photoaffinity labelling experiments by use of a benzoylphenoxy analog of the allylic substrate, farnesyl diphosphate (FPP) indicated that component I participates in binding of FPP through the hydrophobic prenyl tail and that the two components with the allylic substrate form a stable ternary complex.
3) Identification of Active Sites of Prenyltransferases by Genetic Engineering Introduction of many kinds of mutations in the structural genes of FPP synthase of Bacillus stearothermophilus, HepPP synthase of B.subtilis and UPS of M.Iuteus B-P 26 was carried out to find amino acid residues that were important for the
catalyticfunction or substrate binding. Less
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