The Molecular Weight Distribution of Succinoglycan Produced by Sinorhizobium meliloti Is Influenced by Specific Tyrosine Phosphorylation and ATPase Activity of the Cytoplasmic Domain of the ExoP Protein

It is thought that in the gram-negative soil bacterium Sinorhizobium meliloti the protein ExoP is involved in biosynthesis of the acidic exopolysaccharide succinoglycan (EPS I). The amounts and compositions of EPS I produced by mutants expressing ExoP proteins characterized by specific amino acid substitutions in the C-terminal cytoplasmic domain were analyzed. The cytoplasmic domain of the ExoP protein was shown to have ATPase activity. Mutations in the highly conserved Walker A ATP-binding motif prevented ATPase activity of the ExoP protein. Phenotypically, these mutations resulted in much lower levels of succinoglycan which consisted only of monomers of the octasaccharide repeating unit. The ExoP protein has similarities to proteins with autophosphorylating protein tyrosine kinase activity. We found that ExoP was phosphorylated on tyrosine and that site-directed mutagenesis of specific tyrosine residues in the cytoplasmic domain of ExoP resulted in an altered ratio of low-molecular-weight succinoglycan to high-molecular-weight succinoglycan.

据推测，在革兰氏阴性土壤细菌苜蓿中华根瘤菌（Sinorhizobium meliloti）中，蛋白质ExoP参与酸性胞外多糖琥珀酰聚糖（succinoglycan，EPS I）的生物合成。本研究对表达带有C端细胞质结构域特定氨基酸替换的ExoP蛋白的突变株所产生的EPS I的产量与组成进行了分析。研究证实，ExoP蛋白的细胞质结构域具有ATP酶活性。高度保守的Walker A ATP结合基序发生的突变，会使ExoP蛋白丧失ATP酶活性。表型层面上，这类突变会导致琥珀酰聚糖的产量大幅降低，且该多糖仅由八糖重复单元的单体构成。ExoP蛋白与具有自磷酸化蛋白酪氨酸激酶活性的蛋白存在序列相似性。本研究发现，ExoP可在酪氨酸残基上发生磷酸化；对ExoP细胞质结构域中特定酪氨酸残基进行定点诱变，会改变低分子量琥珀酰聚糖与高分子量琥珀酰聚糖的比例。