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

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

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T.Ohshiro, T.Kojima, K.Torii, H.Kawasoe, and Y.Izumi: "Purification and characterization of dibenzothiophene (DBT) sulfone monooxigenase involving in DBT desulfurization of Rhodococcus erythropolis J. Biosci. Biotechnol., 88, 610-616 (1999)"J. Biosci. Bio

T.Ohshiro、T.Kojima、K.Torii、H.Kawasoe 和 Y.Izumi：“涉及红平红球菌 DBT 脱硫的二苯并噻吩 (DBT) 磺单加氧酶的纯化和表征 J. Biosci. Biotechnol., 88, 610-

Yoshikazu Izumi: "Occurrence of bromoperoxidase in the marine green macro-alga, Ulvella lens, and emission of volatile brominated methan by the enzyme"Phytochemistry. 52, 12. 1211-1215 (1999)

Yoshikazu Izumi：“溴过氧化物酶在海洋大型绿藻、石莼晶状体中的出现，以及该酶释放挥发性溴化甲烷的过程”植物化学。

Yoshikazu Izumi: "Characterization, gene cloning and expression of isocitrate lyase involved in the assimilation of one-carbon compounds in an obligate methylotroph, Hyphomicrobium methylovorum GM2"European Journal of Biochemistry. 249, 3. 820-825 (1997)

Yoshikazu Izumi：“专性甲基营养菌、甲基卵微菌 GM2 中参与一碳化合物同化的异柠檬酸裂合酶的表征、基因克隆和表达”《欧洲生物化学杂志》。