Eichhorn E, van der Ploeg J R, Kertesz M A, Leisinger T
Mikrobiologisches Institut, Swiss Federal Institute of Technology, ETH-Zentrum, CH-8092 Zürich, Switzerland.
J Biol Chem. 1997 Sep 12;272(37):23031-6. doi: 10.1074/jbc.272.37.23031.
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., Saller, E., Nashimoto, H., Saito, N., Kertesz, M. A., and Leisinger, T. (1996) J. Bacteriol. 178, 5438-5446). TauD was overexpressed in E. coli to approximately 70% of the total soluble protein and purified to apparent homogeneity by a simple two-step procedure. The apparent Mr of 81,000 of the native protein and the subunit Mr 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 (Km = 55 microM) was the preferred substrate, but pentanesulfonic 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 (Km = 11 microM) supported significant dioxygenase activity.
大肠杆菌的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条件下表达。TauD蛋白与嗜碱假单胞菌的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似性表明，TauD是一种α-酮戊二酸依赖性双加氧酶，催化从牛磺酸中氧解释放亚硫酸盐（van der Ploeg, J. R., Weiss, M. A., Saller, E., Nashimoto, H., Saito, N., Kertesz, M. A., and Leisinger, T. (1996) J. Bacteriol. 178, 5438 - 5446）。TauD在大肠杆菌中过量表达至约占总可溶性蛋白的70%，并通过简单的两步法纯化至表观均一。天然蛋白的表观Mr为81,000，亚基Mr为37,400，与同二聚体结构一致。纯酶将牛磺酸转化为亚硫酸盐和氨基乙醛，氨基乙醛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；亚铁离子是活性绝对必需的；抗坏血酸刺激该反应。因此，这种酶的性质和氨基酸序列将其定义为α-酮戊二酸依赖性双加氧酶家族的一个新成员。纯酶在pH 6.9时显示最大活性，并且在-20℃下储存数周仍保持活性。牛磺酸（Km = 55μM）是首选底物，但戊烷磺酸、3-(N-吗啉代)丙烷磺酸和1,3-二氧代-2-异吲哚啉乙烷磺酸也以显著速率进行脱硫。在所测试的共底物中，只有α-酮戊二酸（Km = 11μM）支持显著的双加氧酶活性。
