Biochemical and Genetic Characterization of PspE and GlpE, Two Single-domain Sulfurtransferases of Escherichia coli.
Biochemical and Genetic Characterization of PspE and GlpE, Two Single-domain Sulfurtransferases of Escherichia coli.
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DOI：
10.2174/1874285800802010018
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发表时间：
2008
期刊：
影响因子：
--
通讯作者：
Larson TJ
中科院分区：
文献类型：
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作者：
Cheng H;Donahue JL;Battle SE;Ray WK;Larson TJ
大肠杆菌的pspE和glpE基因分别编码周质和细胞质单域罗丹斯，催化硫从硫代硫酸盐转移到亲硫受体。构建了其中一种或两种基因缺失的菌株。这些菌株的罗丹斯活性比较表明，PspE提供了85%的罗丹斯活性，GlpE贡献了其余的大部分。在甘油培养基中，PspE的活性是葡萄糖培养基的4倍，并且不受诱导表达psp调控子的条件的影响。glpE/pspE突变体没有表现出明显的生长表型，表明这两个基因都不是生物合成必需含硫分子所必需的。采用阳离子交换色谱法纯化PspE。两个不同的活性峰被洗脱，在稳定共价修饰的程度不同，通过质谱评估。最早的洗脱峰含有两个额外的硫原子，而第二次洗脱峰主要含有一个额外的硫原子。纯化后的PspE的动力学性质与酶硫中间体参与活性位点半胱氨酸的双位移催化机制相一致。SSO32-和CN-的km分别为2.7 mM和32 mM， kcat为64s-1。该酶还催化硫从硫代硫酸盐到二硫代苏糖醇的转移，最终释放硫化物。
The pspE and glpE genes of Escherichia coli encode periplasmic and cytoplasmic single-domain rhodaneses, respectively, that catalyzes sulfur transfer from thiosulfate to thiophilic acceptors. Strains deficient in either or both genes were constructed. Comparison of rhodanese activity in these strains revealed that PspE provides 85% of total rhodanese activity, with GlpE contributing most of the remainder. PspE activity was four times higher during growth on glycerol versus glucose, and was not induced by conditions that induce expression of the psp regulon. The glpE/pspE mutants displayed no apparent growth phenotypes, indicating that neither gene is required for biosynthesis of essential sulfur-containing molecules. PspE was purified by using cation exchange chromatography. Two distinct active peaks were eluted and differed in the degree of stable covalent modification, as assessed by mass spectrometry. The peak eluting earliest contained the equivalent mass of two additional sulfur atoms, whereas the second peak contained mainly one additional sulfur. Kinetic properties of purified PspE were consistent with catalysis occurring via a double-displacement mechanism via an enzyme-sulfur intermediate involving the active site cysteine. Kms for SSO32- and CN- were 2.7 mM and 32 mM, respectively, and kcat was 64s-1. The enzyme also catalyzed transfer of sulfur from thiosulfate to dithiothreitol, ultimately releasing sulfide.