Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus
Characterization and site-directed mutagenesis of Wzb, an O-phosphatase from Lactobacillus rhamnosus
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DOI：
10.1186/1471-2091-9-10
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发表时间：
2008-04-03
期刊：
影响因子：
--
通讯作者：
Gilbert, Christophe
中科院分区：
文献类型：
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作者：
LaPointe, Gisele;Atlan, Daniele;Gilbert, Christophe
背景资料：已提出聚合复合物内的可逆磷酸化事件可以调节肺炎链球菌中的荚膜多糖合成。相似的磷酸酶和激酶基因存在于许多乳酸菌基因组的胞外多糖（EPS）生物合成位点。结果：从鼠李糖乳杆菌ATCC 9595中的wzb基因推导的蛋白质序列揭示了原核O-磷酸酶的聚合酶和组氨醇磷酸酶（PHP）超家族的四个基序。从大肠杆菌培养物中纯化天然和修饰的His-标签融合Wzb蛋白。提取物对含酪氨酸的肽表现出磷酸酶活性。纯化的融合蛋白Wzb对pNPP有活性，在pH7.3和75 ℃下，在1%牛血清白蛋白（BSA）存在下具有最佳活性。在50 ℃时，残余活性降至10%.铜离子是磷酸酶活性所必需的，这是显着增加，通过添加钴。突变的融合Wzb蛋白表现出降低的磷酸酶活性对硝基苯磷酸。然而，一个变体（C6 S）表现出接近20%的磷酸酶activity.Conclusion增加：这些特点揭示了显着差异与锰依赖CpsB蛋白酪氨酸磷酸酶描述的肺炎链球菌，以及与多糖相关的磷酸酶的革兰氏阴性菌。
Background: Reversible phosphorylation events within a polymerisation complex have been proposed to modulate capsular polysaccharide synthesis in Streptococcus pneumoniae. Similar phosphatase and kinase genes are present in the exopolysaccharide (EPS) biosynthesis loci of numerous lactic acid bacteria genomes.Results: The protein sequence deduced from the wzb gene in Lactobacillus rhamnosus ATCC 9595 reveals four motifs of the polymerase and histidinol phosphatase (PHP) superfamily of prokaryotic O-phosphatases. Native and modified His-tag fusion Wzb proteins were purified from Escherichia coli cultures. Extracts showed phosphatase activity towards tyrosine-containing peptides. The purified fusion protein Wzb was active on p-nitrophenyl-phosphate (pNPP), with an optimal activity in presence of bovine serum albumin (BSA 1%) at pH 7.3 and a temperature of 75 C. At 50 C, residual activity decreased to 10 %. Copper ions were essential for phosphatase activity, which was significantly increased by addition of cobalt. Mutated fusion Wzb proteins exhibited reduced phosphatase activity on p-nitrophenyl-phosphate. However, one variant (C6S) showed close to 20% increase in phosphatase activity.Conclusion: These characteristics reveal significant differences with the manganese-dependent CpsB protein tyrosine phosphatase described for Streptococcus pneumoniae as well as with the polysaccharide-related phosphatases of Gram negative bacteria.