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

• 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
• 来源: https://kaken.nii.ac.jp/grant/KAKENHI-PROJECT-09044224/
• 关键词: C1 microorganism; serine pathway; marine algae; bpo gene; stereoselective synthesis; isocitrate lyase; serine-glyoxylate aminotransferase; hydroxypyruvate reductase; ヒドロキシピルビン酸レタクターゼ; 結晶化; 基質-補酵素-酸素複合体; メチロトーフ; Hyphomicrobium methylovorum; セリ; C1 microorganism; serine pathway; marine algae; bpo gene; stereoselective synthesis; isocitrate lyase; serine-glyoxylate aminotransferase; hydroxypyruvate reductase; ヒドロキシピルビン酸レタクターゼ; 結晶化; 基質-補酵素-酸素複合体; メチロトーフ; Hyphomicrobium methylovorum; セリ

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

这项研究的研究结果总结如下。（1）针对C1微生物的新型酶的大规模制备和一般表征：一种新型酶等异源性裂解液酶（ICL），来自C1微生物，菌丝菌丝菌丝体，我们从甲基苯甲酸和甲基苯甲酸中分离出来，并纯化了甲基化的甲基甲基化酶，并被纯化。重新检查了菌粒菌株中酶活性的分布，从而证明在所有测试的菌株中，酶活性或多或少都与报道的结果不同。 Then, the ICL of Hyphomicrobium methylovorum was purified to homogeneity.. A large quantity of serine-glyoxylate aminotransferase (SGAT) was also purified to homogeneity from E.coli pKK223-3/SGAT which carried a higher-perexpressing vector with the SGAT gene of Hyphomicrobium methylovorum.(2)Crystallation of针对C1微生物和X射线晶体学分析的新型酶：至于羟型扭转酸酯还原……更多（HPR），一种全酶，具有辅酶NAD（双锥体形式）的全酶（HPR），根据Apo-enzyme的结晶过程，我们已经建立了apo-Enzyme。由于全酶晶体的X射线晶体学的结果，发现NAD正如我们预期的那样与酶位点结合。此外，与我们期望的是，催化位点和脱酶的底物结合位点之间的裂纹角度必须发生变化，而与Apo-enzyme相比，全酶的结构是没有塑料的。我们成功地获得了酶的三元络合物（底物 - alaalog-NAD和酶）的结晶，因此目前正在进行X射线晶体学分析。至于SGAT，我们通过使用与晶体屏幕的悬挂滴法获得了菱形板形式的晶体。酶通过不同的反应机理催化反应，形成了其他已知的氨基转移酶。