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

本研究的研究成果归纳如下。（1）C1微生物特异性新酶的大规模制备和一般表征：从C1微生物Hyphomicrobium methylovorum中分离到一种新的异柠檬酸裂解酶（ICL），其是从甲醇中分离丝氨酸的最佳生产者。重新检查了丝微菌属菌株中酶活性的分布，结果表明，与报道的结果不同，在所有测试菌株中或多或少都发现了酶活性。然后，将菌丝微菌的ICL纯化至均一。同时，从携带食甲基丝微菌SGAT基因的大肠杆菌pKK 223 -3/SGAT中纯化得到了大量的SGAT。（2）C1微生物特异性新酶的结晶和X射线晶体学分析： ...更多信息 (HPR)根据我们已经建立的脱辅基酶的结晶方法，使具有辅酶NAD的全酶结晶（双锥体形式）。作为全酶晶体的X射线晶体学的结果，发现NAD如我们预期的那样结合到酶的位点。此外，与我们预期的脱辅基酶的催化位点和底物结合位点之间的裂缝角度必须有变化相反，与脱辅基酶相比，全酶的结构没有改变。我们成功地获得了酶的三元复合物（底物-丙氨酸-NAD和酶）的结晶，因此X射线晶体学分析正在进行中。对于SGAT，我们用悬滴法用晶体筛Ⅰ获得了菱形片状晶体。（3）一种新的C1微生物特异性酶SGAT的动力学研究：对于SGAT，我们与Coolk教授的研究小组合作，采用停流法和同位素标记底物进行了动力学研究，发现该酶通过与其他已知氨基转移酶不同的反应机理催化该反应。少

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