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Comparative biochemical studies on structure, function and physiology of catechol dioxygenases

儿茶酚双加氧酶结构、功能和生理学的比较生化研究

基本信息

批准号：
02454565
负责人：
NOZAKI Mitsuhiro
金额：
$ 0.64万
依托单位：
Shiga University of Medical Science
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1991
项目状态：
已结题

项目摘要

Catechol dioxygenases are classified into two groups : intradiol and extradiol. We have previously determined the DNA sequence of an extradiol enzyme, catechol 2, 3-dioxygenase. In order to pursue comparative studies between various catechol dioxygenases, we have tried to determine the DNA sequence of intradiol enzymes.1. we have studied the well known intradiol enzyme, catechol 1, 2dioxygenase and found for the first time the existence of 3 isozymes, au, ap, op, of this enzyme from Pseudomonas arvilla C-1. we also succeeded to clone the gene for this enzyme from Pseudomonas putida mt-2.2. An enzyme which cleaves the benzene ring of 3, 5-dichlorocatechol has been purified to homogeneity for the first time from a Pseudoiwnad, grown with a herbicide, 2, 4-dichlorophenoxyacetic acid (2, 4-D), as the sole carbon source. The enzyme was found to be a nonheme ferric dioxygenase and to catalyze the intradiol cleavage of 3, 5-dichlorocatechol. Thus, the enzyme was designated as 3, 5-dichlorocatechol 1, 2dioxygenase. Judging from the substrate specificity, kinetic constants, and N-terminal amino acid sequence, the enzyme was distinct from other chlorocatechol dioxygenases reported previously. We have also succeeded to clone the gene for this enzyme and the determination of the DNA sequence is now underway.3. For the elucidation of the active site structure of catechol 2, 3dioxygenase, we used o-nitrophenol, a competitive inhibitor, and found it a useful active site probe for this enzyme. Using this probe, a novel mode interaction between substrate or the analog and the enzyme has been proposed. Trials were made to substitute the iron cofactor in the active site of this enzyme by other metals, and to modify the enzyme by sulfhydryl (-SH) blocking reagents or by suicide substrata. Basic information has been obtained from these studies on this enzyme.

儿茶酚双加氧酶分为两类：内源性和外源性。我们之前已经测定了一种外源酶，儿茶酚2，3-双加氧酶的DNA序列。为了进行不同儿茶酚双加氧酶的比较研究，我们尝试测定了吲哚酚双加氧酶的DNA序列。我们研究了已知的儿茶酚-1，2-双加氧酶，首次从假单胞菌C-1中发现了该酶的Au、AP、OP三种同工酶。我们还成功地从恶臭假单胞菌MT-2.2中克隆了该酶的基因。以除草剂2，4-二氯苯氧基乙酸(2，4-D)为唯一碳源生长的假单胞菌，首次纯化得到一种裂解3，5-二氯邻苯二酚苯环的酶。该酶是一种非血红素铁双加氧酶，催化3，5-二氯儿茶酚的传统裂解。因此，该酶被命名为3，5-二氯邻苯二酚1，2双加氧酶。从底物专一性、动力学常数和N端氨基酸序列来看，该酶不同于以往报道的其他氯代儿茶酚双加氧酶。我们还成功地克隆了该酶的基因，DNA序列的测定正在进行中。为了阐明邻苯二酚2，3双加氧酶的活性部位结构，我们使用了竞争性抑制剂邻硝基苯酚，并发现它是该酶的一个有用的活性部位探针。利用该探针，提出了底物或类似物与酶的一种新的相互作用模式。有人尝试用其他金属取代该酶活性部位的铁辅因子，并用-SH封闭剂或自杀底物修饰该酶。从这些研究中已经获得了关于这种酶的基本信息。

项目成果

期刊论文数量（32）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

S,Yamamoto,M.Nozaki,and Y.Ishimura(editors): "International Symposium on Oxygenases and Oxygen Activation" Yamada Science Foundation, 217 (1991)

S，Yamamoto，M.Nozaki，和 Y.Ishimura（编辑）：“国际氧化酶和氧活化研讨会”山田科学基金会，217（1991）

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H.Tojo,K.Horiike,T.Ishida,T.Kobayashi,M.Nozaki and M.Okamoto: "Analytical and microーpreparative highーperformance gel chromatography of proteins with a short column of porous silicaーbased aqueous gels:Estimation of molecular size rapid determination of lig

H. Tojo、K. Horiike、T. Ishida、T. Kobayashi、M. Nozaki 和 M. Okamoto：“使用短柱多孔二氧化硅水凝胶对蛋白质进行分析和微制备高性能凝胶色谱：估计分子大小的快速测定

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M.Nozaki,C.Nakai,K.Horiike,S.Kuramitsu,and H.Kagamiyama(edited by C.C.Reddy,G.A.Hamilton and K.M.Medyastha): "Three isozymes of catechol 1,2-dioxygenase (pyrocatechase) from Pseudomonas arvilla Cー1." Biological Oxidation Systems. 1. 405-416 (1990)

M. Nozaki、C. Nakai、K. Horiike、S. Kuramitsu 和 H. Kagamiyama（由 C. C. Reddy、G. A. Hamilton 和 K. M. Medyastha 编辑）：“来自 Pseudomonas arvilla C 的儿茶酚 1,2-双加氧酶（焦儿茶酶）的三种同工酶-1.”生物氧化系统。1. 405-416 (1990)