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

本研究的主要研究成果如下：(1)新型C1族微生物专一性酶的大规模制备及一般性质研究：从C1型微生物中分离到一种新的异柠檬酸裂解酶(ICL)，该酶是我们从甲醇中筛选到的丝氨酸最佳产生菌，对其进行了纯化和性质研究。结果表明，所有供试菌株都或多或少地发现了酶活性，与文献报道的结果不同。在此基础上，对甲基亚微生物菌的ICL进行纯化，得到均一的ICL。从大肠杆菌pKK223-3/sGat中也纯化到了大量的丝氨酸乙醛酸氨基转移酶(Sgat)。(2)新型C1微生物专一性酶的结晶和X射线晶体分析：羟基丙酮酸还原酶…More(HPR)是一种含有辅酶NAD的全酶，按照我们已经建立的脱辅酶的结晶程序进行结晶(双锥体形式)。对全酶晶体进行X射线结晶学分析，发现NAD如我们所预期的那样结合在酶的位置上。此外，与我们预期的脱辅酶的催化位置和底物结合位置之间的夹角必须改变相反，全酶的结构与脱辅酶的结构不同。我们成功地获得了该酶(底物-丙二醛-NAD和酶)三元络合物的结晶，因此X射线晶体分析正在进行中。对于SGAT，我们用晶体筛I的悬滴法得到了菱形的板状晶体。(3)一种新的针对C1微生物的酶SGAT的动力学研究：关于SGAT，我们与Coolk教授的团队合作，用停流法和使用同位素标记的底物进行了动力学研究，结果表明该酶通过与其他已知的氨基转移酶不同的反应机制催化了该反应。较少

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