Characterization of alpha-ketoglutarate-dependent taurine dioxygenase from Escherichia coli
Characterization of alpha-ketoglutarate-dependent taurine dioxygenase from Escherichia coli
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DOI：
10.1074/jbc.272.37.23031
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发表时间：
1997-09-12
影响因子：
4.8
通讯作者：
Leisinger, T
中科院分区：
文献类型：
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作者：
Eichhorn, E;vanderPloeg, JR;Leisinger, T
大肠杆菌tauD基因是利用牛磺酸（2-氨基乙磺酸）作为硫源所必需的，并且仅在硫酸盐饥饿的条件下表达。TauD蛋白与真养产碱杆菌的α-酮戊二酸依赖性2，4-二氯苯氧基乙酸双加氧酶的序列相关性表明，TauD是催化从牛磺酸氧解释放亚硫酸盐的α-酮戊二酸依赖性双加氧酶（货车der Ploeg，J.R.，韦斯，M.一、赛勒，E.. Nashimoto，H.，Saito，N.，Kertesz，M.一、和Leisinger，T.等人（1996）J. Bacteriol. 178，5438-5446）。TauD在E.大肠杆菌中的可溶性蛋白的含量约为总可溶性蛋白的70%，并通过简单的两步程序纯化至表观均一性。天然蛋白质的表观M-r为81，000，亚基M-r为37，400，与同源二聚体结构一致。纯酶将牛磺酸转化为亚硫酸盐和氨基乙醛，其在酶促转化为乙醇胺后通过高压液相色谱法鉴定。该反应还消耗等摩尔量的氧气和α-酮戊二酸;亚铁是绝对需要的活动;和抗坏血酸刺激反应。该酶的性质和氨基酸序列因此将其定义为α-酮戊二酸依赖性双加氧酶家族的新成员。纯酶在pH6.9时显示最大活性，并在-20 ℃下储存数周后仍保持活性。牛磺酸（K-m = 55 μ M）是优选的底物，但戊烷磺酸、3-（N-吗啉代）丙磺酸和1，3-二氧代-2-异吲哚啉乙磺酸也以显著的速率被磺化。在测试的共底物中，只有α-酮戊二酸（Km = 11 μ M）支持显着的双加氧酶活性。
The Escherichia coli tauD gene is required for the utilization of taurine (2-aminoethanesulfonic acid) as a sulfur source and is expressed only under conditions of sulfate starvation. The sequence relatedness of the TauD protein to the alpha-ketoglutarate-dependent 2,4-dichlorophenoxyacetate dioxygenase of Alcaligenes eutrophus suggested that TauD is an alpha-ketoglutarate-dependent dioxygenase catalyzing the oxygenolytic release of sulfite from taurine (van der Ploeg, J. R., Weiss, M. A., Sailer, E.. Nashimoto, H., Saito, N., Kertesz, M. A., and Leisinger, T. (1996) J. Bacteriol. 178, 5438-5446). TauD was overexpressed in E. coli to similar to 70% of the total soluble protein and purified to apparent homogeneity by a simple two-step procedure. The apparent M-r of 81,000 of the native protein and the subunit M-r of 37,400 were consistent with a homodimeric structure. The pure enzyme converted taurine to sulfite and aminoacetaldehyde, which was identified by high pressure liquid chromatography after enzymatic conversion to ethanolamine. The reaction also consumed equimolar amounts of oxygen and alpha-ketoglutarate; ferrous iron was absolutely required for activity; and ascorbate stimulated the reaction. The properties and amino acid sequence of this enzyme thus define it as a new member of the alpha-ketoglutarate-dependent dioxygenase family. The pure enzyme showed maximal activity at pH 6.9 and retained activity on storage at -20 degrees C for several weeks. Taurine (K-m = 55 mu M) was the preferred substrate, but pentane-sulfonic acid, 3-(N-morpholino)propanesulfonic acid, and 1,3-dioxo-2-isoindolineethanesulfonic acid were also desulfonated at significant rates. Among the cosubstrates tested, only alpha-ketoglutarate (K-m = 11 mu M) supported significant dioxygenase activity.