Elucidation of Structure and Reaction Mechanism of Novel Crystalline Enzymes Specific for C1 Microoranisms

C1微生物特异性新型结晶酶的结构和反应机制的阐明

基本信息

批准号：
09044224
负责人：
IZUMI Yoshikazu
金额：
$ 9.02万
依托单位：
Tottori University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A).
财政年份：
1997
资助国家：
日本
起止时间：
1997 至 1999
项目状态：
已结题

项目摘要

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 … More (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

本研究的研究结果总结如下：(1)C1微生物特异性新型酶的大规模制备和一般表征：从C1微生物甲基丝微生物中分离出的一种新型酶异柠檬酸裂解酶(ICL)，我们将其作为最好的生产者分离出来。从甲醇中纯化丝氨酸，并对其酶活性在丝菌属菌株中的分布进行了重新检查，结果表明酶活性大于或等于。与报道的结果不同，在所有测试的菌株中发现的结果较少。然后，将甲基丝菌的 ICL 纯化至均质。还从大肠杆菌 pKK223-3/SGAT 中纯化至均质的大量丝氨酸乙醛酸转氨酶（SGAT）。其携带甲基丝微菌SGAT基因超表达载体。(2)新型酶的结晶C1微生物和X射线晶体分析：对于羟基丙酮酸还原酶（HPR），根据我们已经建立的脱辅基酶的结晶程序，将具有辅酶NAD的全酶结晶（双锥体形式）。作为全酶晶体的 X 射线晶体学结果，我们发现 NAD 与酶的位点结合。此外，与我们预期脱辅基酶的催化位点和底物结合位点之间的裂口角度一定有变化相反，全酶的结构与脱辅基酶的结构相比没有改变。我们成功获得了酶的三元复合物（底物-类似物-NAD和酶）的结晶，因此对于SGAT，我们正在进行X射线晶体学分析。通过使用 Crystal Screen I 的悬滴法，获得了菱形板状晶体。(3)C1 微生物特有的新型酶 SGAT 的动力学研究：对于 SGAT，通过与 Coolk 教授的小组合作进行动力学研究通过停流法并使用同位素标记的底物进行了研究，结果表明该酶通过与其他已知转氨酶不同的反应机制催化反应。