Elucidation of regulation mechanism of sphingolipid biosynthesis and degradation based on protein structure.
| Project/Area Number |
18570114
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Structural biochemistry
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| Research Institution | Osaka Medical College |
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Principal Investigator |
IKUSHIRO Hiroko Osaka Medical College, Faculty of Medicine, Junior Associate Professor (10280702)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥4,110,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥510,000)
Fiscal Year 2007: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
Fiscal Year 2006: ¥1,900,000 (Direct Cost: ¥1,900,000)
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| Keywords | sphingolipid / serine palmitoyltransferase / enzyme reaction mechanism / protein structural analysis / enzyme / lipid / protein / biomolecule / 立体結晶構造解析 / PLP酵素 |
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| Research Abstract |
Serine palmitoyltransferase (SPT) catalyzes the first and rate-limiting step reaction of the sphingolipid biosynthesis. The change of the SPT activity influences directly the total amount and the kind of the cellar sphingolipids. Therefore, the elucidation of the regulation mechanism of SPT is very important. (1) We cloned the SPT genes of the various kinds of the sphingolipid-content bacteria, constructed the overexpression system of SPT in E. coli, and established the purification method of each recombinant SPT. (2) We succeeded in synthesis of a non-reactive analogue of palmitoyl-CoA, S-(2-oxoheptadecyl)-CoA. Using the purified recombinant SPT, the reaction of SPT with L-serine and S-(2-oxoheptadecyl)-CoA was examined in detail. By the addition of L-serin to SPT, the external aldimine intermediate is formed via the Michaelis complex. When S-(2-oxoheptadecyl)-CoA was added to this SPT-L-serine binary complex, it was not converted to the final product 3-ketodihydrosphingosine, but the
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new absorption peak of the quinonoid intermediate was detected. The kinetic analysis of time resolved spectra of this ternary complex showed that the binding of the S-(2-oxoheptadecyl)-CoA to the SPT induces the formation. And accumulation of the quinonoid intermediate and the SPT reaction is in equilibrium between the quinonid intermediate and the former external aldimine intermediate. (3) The rate of the α-deprotonation of L-serin by SPT, that corresponds to the rate of the quinonoid formation, was examined by NMR. NMR studies showed that the hydrogen-deuterium exchange at Ca of L-serine is very slow in the SPT-L-serine external aldimine complex, but the rate is 100-fold increased by the addition of S-(2-oxoheptadecyl)-CoA, showing a remarkable substrate synergism in SPT. (4) We succeeded in crystallization of SPT. The structure of SPT・L-serine external aldimine complex had been determined. Based on the crystallographic data, the discussion about the role of the conserved amino acid residues in the SPT catalysis became possible. Less
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Report
(3 results)
Research Products
(38 results)
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[Book] Structural Biology of Sphingolipid Synthesis. in Sphingolipid Biology (Hirabayashi, Y., Igarashi, Y., Merrill, A., Eds.)2006
Author(s)
Ikushiro, H., Okamoto, A., Hayashi H.
Total Pages
10
Publisher
Springer-Verlag Tokyo/Japan (pp.483-492(2006))
Related Report
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