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首页> 外文期刊>Journal of bacteriology >Biochemical and Genetic Evidence formeta-Ring Cleavage of 2,4,5-Trihydroxytoluene inBurkholderia sp. Strain DNT
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Biochemical and Genetic Evidence formeta-Ring Cleavage of 2,4,5-Trihydroxytoluene inBurkholderia sp. Strain DNT

机译：伯克霍尔德菌中2,4,5-三羟基甲苯的生化和遗传证据形成环裂解。应变DNT

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2,4,5-Trihydroxytoluene (THT) oxygenase fromBurkholderia sp. strain DNT catalyzes the conversion of THT to an unstable ring fission product. Biochemical and genetic studies of THT oxygenase were undertaken to elucidate the mechanism of the ring fission reaction. The THT oxygenase gene (dntD) was previously localized to the 1.2-kb DNA insert subcloned in the recombinant plasmid designated pJS76 (W. C. Suen and J. C. Spain, J. Bacteriol. 175:1831–1837, 1993). Analysis of the deduced amino acid sequence of DntD revealed the presence of the highly conserved residues characteristic of the catechol 2,3-dioxygenase gene family I. The deduced amino acid sequence of DntD corresponded to a molecular mass of 35 kDa. The native molecular masses for the THT oxygenase estimated by using gel filtration chromatography and nondenaturing gel electrophoresis were 67.4 and 77.8 kDa, respectively. The results suggested that the native protein consists of two identical subunits. The colorless protein contained 2 mol of iron per mol of protein. Stimulation of activity in the presence of ferrous iron and ascorbate suggested a requirement for ferrous iron in the active site. The properties of the enzyme are similar to those of the catechol 2,3-dioxygenases (meta-cleavage dioxygenases). In addition to THT, the enzyme exhibited activity towards 1,2,4-benzenetriol, catechol, 3- and 4-methylcatechol, and 3- and 4-chlorocatechol. The chemical analysis of the THT ring cleavage product showed that the product was 2,4-dihydroxy-5-methyl-6-oxo-2,4-hexadienoic acid, consistent with extradiol ring fission of THT.
机译：来自伯克霍尔德菌 sp。的2,4,5-三羟基甲苯（THT）加氧酶。 DNT菌株催化THT转化为不稳定的环裂变产物。进行了THT加氧酶的生化和遗传研究，以阐明环裂变反应的机理。 THT加氧酶基因（ dntD ）先前位于亚克隆到名为pJS76的重组质粒中的1.2 kb DNA插入片段中（WC Suen和JC Spain，J. Bacteriol。175：1831-1837，1993）。 。对推导的DntD氨基酸序列的分析表明，存在儿茶酚2，3-二加氧酶基因家族I特征的高度保守的残基。推导的DntD氨基酸序列对应于35kDa的分子量。通过使用凝胶过滤色谱法和非变性凝胶电泳估计的THT加氧酶的天然分子量分别为67.4和77.8 kDa。结果表明天然蛋白质由两个相同的亚基组成。无色蛋白质每摩尔蛋白质包含2摩尔铁。在亚铁和抗坏血酸存在下刺激活性表明在活性部位需要亚铁。该酶的性质与邻苯二酚2,3-双加氧酶（ meta -裂解双加氧酶）相似。除THT以外，该酶还对1,2,4-苯三酚，邻苯二酚，3-和4-甲基邻苯二酚以及3-和4-氯邻苯二酚具有活性。 THT环裂解产物的化学分析表明，该产物为2，4-二羟基-5-甲基-6-氧代-2，4-己二酸，与THT的二醇外环裂一致。

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