1999 Fiscal Year Final Research Report Summary
Elucidation of Structure and Reaction Mechanism of Novel Crystalline Enzymes Specific for C1 Microoranisms
| Project/Area Number |
09044224
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| Research Category |
Grant-in-Aid for Scientific Research (A).
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
応用微生物学・応用生物化学
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| Research Institution | Tottori University |
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Principal Investigator |
IZUMI Yoshikazu Tottori University, Faculty of Engineering, Professor, 工学部, 教授 (40026555)
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| Co-Investigator(Kenkyū-buntansha) |
NAGASAWA Toru Gifu University, Faculty of Engineering, Professor, 工学部, 教授 (60115904)
OGATA Seiya Kyushu University, Faculty of Agriculture, Professor, 農学部, 教授 (20038277)
OHSHIRO Takashi Tottori University, Faculty of Engineering, Research Associate, 工学部, 助手 (00233106)
TANABE Tadashi Research Institute of National Cardiovasucular Center, Department Director, 薬理部, 部長 (60025624)
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| Project Period (FY) |
1997 – 1999
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| Keywords | C1 microorganism / serine pathway / marine algae / bpo gene / stereoselective synthesis / isocitrate lyase / serine-glyoxylate aminotransferase / hydroxypyruvate reductase |
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| Research Abstract |
The research results of this study are summarized as follows.
(1)Large scale preparation and general characterization of novel enzymes specific for C1 microorganisms : A novel enzyme isocitrate lyase (ICL) from the C1 microorganism, Hyphomicrobium methylovorum which we isolated as the best producer of serine from methanol, was purified and characterized. The distribution of the enzyme activities in Hyphomicrobium strains was reexamined, resulting in demonstrating that the enzyme activities were more or less found in all the strains tested unlike the reported results. Then, the ICL of Hyphomicrobium methylovorum was purified to homogeneity.. A large quantity of serine-glyoxylate aminotransferease (SGAT) was also purified to homogeneity from E.coli pKK223-3/SGAT which carried a highperexpressing vectore with the SGAT gene of Hyphomicrobium methylovorum.
(2)Crystallization of novel enzymes specific for C1 microorganisms and X-ray crystallographic analyses : As for hydroxypyruvate reductase
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(HPR), a holo-enzyme which had the coenzyme NAD was crystallized (bipyramidal form) according to the crystallization procedures of the apo-enzyme which we have already established. As a result of X-ray crystallography of the holo-enzyme crystals, NAD was found to be bound to the site of the enzyme as we expected. Moreover, on the contrary to our expectation that there must be a change in angle of the cleft between the catalytic site and substrate binding site of the apo-enzyme, the structure of the holo-enzyme was unchaged as compared to that of the apo-enzyme. We succeeded in obtaining crystallization of the ternary complex of the enzyme (substrate-alalog-NAD and enzyme), so X-ray crystallographic analyses are now underway. As for the SGAT, we have obtained crystals of rhombus plate form by using the hanging drop method with Crystal Screen I.
(3)kinetic studies of a novel enzyme, SGAT, specific for C1 microorganisms : As for SGAT, by the collaboration with Prof. Coolk's group, kinetic studies were carried out by the stopped-flow method and by use of the isotope-labelled substrates, and revealed that the enzyme catalyzed the reaction through a different reaction mechanism form other known aminotransfereases. Less
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