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

儿茶酚双加氧酶分为两类：内二醇和外二醇。我们以前已经确定了一个额外的二醇酶，儿茶酚2，3-双加氧酶的DNA序列。为了进行各种儿茶酚双加氧酶之间的比较研究，我们试图确定内二醇酶的DNA序列。我们研究了已知的内二醇酶--邻苯二酚1，2-双加氧酶，并首次发现存在3种同工酶，Au，ap，op。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。从以除草剂2，4-二氯苯氧乙酸（2，4-D）为唯一碳源生长的假丝酵母（Pseudoiwnad）中首次纯化出一种能裂解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3，5-二氯邻苯二酚的二醇内裂解。因此，将该酶命名为3，5-二氯儿茶酚1，2-双加氧酶。从底物特异性、动力学常数和N-末端氨基酸序列来看，该酶不同于以往报道的其他氯儿茶酚双加氧酶。我们还成功地克隆了这种酶的基因，DNA序列的测定正在进行中。为了阐明邻苯二酚2，3双加氧酶的活性中心结构，我们用邻硝基苯酚作为竞争性抑制剂，发现邻硝基苯酚是一种有用的邻苯二酚2，3双加氧酶活性中心探针。利用这种探针，底物或类似物与酶之间的一种新的相互作用模式已被提出。尝试用其他金属取代该酶活性部位的铁辅因子，并用巯基（-SH）封闭剂或自杀底物修饰该酶。从这些研究中获得了关于这种酶的基本信息。

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.