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

本研究的研究结果总结如下：(1) C1微生物专用新型酶的大规模制备和一般表征：从C1微生物中分离到的产丝氨酸能力最强的菌丝微生物（Hyphomicrobium methylovorum）中分离到一种新型的异柠檬酸裂解酶（ICL），并对其进行了纯化和表征。对菌丝微生物菌株的酶活性分布进行了重新检查，结果表明，与报道的结果不同，所有菌株的酶活性或多或少都存在。然后，对甲基化丝孢菌的ICL进行纯化，达到均匀性。从大肠杆菌pKK223-3/SGAT中纯化出大量的丝氨酸-乙氧基氨基转移酶（SGAT），该酶携带甲基化丝孢菌SGAT基因的高表达载体。(2) C1微生物专用新型酶的结晶和x射线晶体学分析：对于羟丙酮酸还原酶（HPR），根据我们已经建立的载子酶的结晶程序，结晶了一种具有辅酶NAD的全酶（双锥体形式）。通过对全酶晶体的x射线结晶学分析，发现NAD与酶的位点相结合。此外，与我们的预期相反，载脂蛋白酶的催化位点和底物结合位点之间的间隙角度必须发生变化，与载脂蛋白酶相比，holo酶的结构没有改变。我们成功地获得了酶的三元配合物（底物-alalog- nad和酶）的结晶，因此x射线晶体学分析正在进行中。SGAT,我们获得的菱形晶体板形式通过悬滴法与水晶屏幕。(3)动力学研究的新酶,SGAT,具体为C1微生物:至于SGAT,与Coolk教授合作的团体,动力学研究是由stopped-flow isotope-labelled基质的方法和利用,并透露,酶催化反应形成其他已知的aminotransfereases通过不同的反应机制。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