Elucidation of Structure and Reaction Mechanism of Novel Crystalline Enzymes Specific for C1 Microoranisms
Elucidation of Structure and Reaction Mechanism of Novel Crystalline Enzymes Specific for C1 Microoranisms
批准号:
09044224
负责人:
IZUMI Yoshikazu
金额:
$9.02万
依托单位:
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
项目状态:
已结题
起止时间:
1997 至 1999
中文摘要
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
英文摘要
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
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Tadashi Tanabe: "Inverse gene expression of prostacyclin and thromboxane synthascs in resident and activated peritoncal macrophages" FEBS Letters. 409,2. 242-246 (1997)
Tadashi Tanabe:“驻留和激活的腹膜巨噬细胞中前列环素和血栓素合成酶的反向基因表达”FEBS Letters。
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Peter Brock: "Structural basis for the activation of phenylalanine in the non-ribosomal biosynthesis of gramicidin S" EMBO Jorunal. 16,14. 4174-4183 (1997)
Peter Brock:“短杆菌肽 S 非核糖体生物合成中苯丙氨酸激活的结构基础”EMBO Jorunal。
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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-
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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。
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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》。
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共 18 条
Studies on the structure and function of enzymes related to C-S bond formation and cleavage of useful naturally-occuring cyclic compounds having sulfur
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批准号:21580093
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项目类别:Grant-in-Aid for Scientific Research (C)
-
资助金额:$3.16万
-
财政年份:2009
-
负责人:IZUMI Yoshikazu
-
依托单位:
Improvement of Functions of Novel Enzymes in the Desulfurization Metaoblism of Petroleum by Protein Engineering and Molecular Genetics
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批准号:15580063
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项目类别:Grant-in-Aid for Scientific Research (C)
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资助金额:$2.43万
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财政年份:2003
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负责人:IZUMI Yoshikazu
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依托单位:
Studies on Distribution of Marine Macro-algae in Europe Which Produce Novel Useful Enzymes and Their Structure-Function
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批准号:15404025
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项目类别:Grant-in-Aid for Scientific Research (B)
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资助金额:$7.87万
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财政年份:2003
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负责人:IZUMI Yoshikazu
-
依托单位:
Elucidation of properties of novel enzymes catalyzing the formation and the cleavage of carbon-sulfur bond in microooganisms
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批准号:11660091
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项目类别:Grant-in-Aid for Scientific Research (C)
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资助金额:$1.98万
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财政年份:1999
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负责人:IZUMI Yoshikazu
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依托单位:
Elucidation of functions of novel enzymes catalyzing the formation and the cleavage of cabond and their application
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批准号:09650877
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项目类别:Grant-in-Aid for Scientific Research (C)
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资助金额:$2.05万
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财政年份:1997
-
负责人:IZUMI Yoshikazu
-
依托单位:
Development of production process for physiologically active substanses using haenzymes from marine alga and microoganism
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批准号:07556092
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项目类别:Grant-in-Aid for Scientific Research (A)
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资助金额:$3.01万
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财政年份:1995
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负责人:IZUMI Yoshikazu
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依托单位:
Enzymatic and Molecular Genetic Studies of Serine Production by C_1-Microorganisms
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批准号:06650917
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项目类别:Grant-in-Aid for General Scientific Research (C)
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资助金额:$1.28万
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财政年份:1994
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负责人:IZUMI Yoshikazu
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依托单位:
Studies on the Metabolic Enzymes Characteristic to C_1-Microorganisms----Structures and Functions
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批准号:06044148
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项目类别:Grant-in-Aid for international Scientific Research
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资助金额:$3.14万
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财政年份:1994
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负责人:IZUMI Yoshikazu
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依托单位:
Halogenating Enzymes Produced by Marine Algae and Microbes---Structures and Functions
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批准号:04044118
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项目类别:Grant-in-Aid for international Scientific Research
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资助金额:$8.13万
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财政年份:1992
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负责人:IZUMI Yoshikazu
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依托单位:
Enzymzyic znd Molecular Genetic Studies of Serine Production by C_1-Microorganisms
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批准号:04660119
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项目类别:Grant-in-Aid for General Scientific Research (C)
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资助金额:$1.22万
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财政年份:1992
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负责人:IZUMI Yoshikazu
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依托单位:
Studies on the Characterization of Metabolic Properties of C_1-microorganisms and Its Application to Serine Synthesis
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批准号:01560121
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项目类别:Grant-in-Aid for General Scientific Research (C)
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资助金额:$1.22万
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财政年份:1989
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负责人:IZUMI Yoshikazu
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依托单位: