Laboratory of Basic and Applied Molecular Biotechnology, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto, Japan.
Laboratory of Food Microbiology, Department of Life Science, Faculty of Science and Engineering, Setsunan University, Neyagawa, Osaka, Japan.
Biosci Biotechnol Biochem. 2021 Nov 24;85(12):2410-2419. doi: 10.1093/bbb/zbab170.
Gram-negative Sphingomonas sp. A1 incorporates acidic polysaccharide alginate into the cytoplasm via a cell-surface alginate-binding protein (AlgQ2)-dependent ATP-binding cassette transporter (AlgM1M2SS). We investigated the function of calcium bound to the EF-hand-like motif in AlgQ2 by introducing mutations at the calcium-binding site. The X-ray crystallography of the AlgQ2 mutant (D179A/E180A) demonstrated the absence of calcium binding and significant disorder of the EF-hand-like motif. Distinct from the wild-type AlgQ2, the mutant was quite unstable at temperature of strain A1 growth, although unsaturated alginate oligosaccharides stabilized the mutant by formation of substrate/protein complex. In the assay of ATPase and alginate transport by AlgM1M2SS reconstructed in the liposome, the wild-type and mutant AlgQ2 induced AlgM1M2SS ATPase activity in the presence of unsaturated alginate tetrasaccharide. These results indicate that the calcium bound to EF-hand-like motif stabilizes the substrate-unbound AlgQ2 but is not required for the complexation of substrate-bound AlgQ2 and AlgM1M2SS.
革兰氏阴性鞘氨醇单胞菌 A1 通过细胞表面的褐藻胶结合蛋白（AlgQ2）依赖性 ATP 结合盒转运蛋白（AlgM1M2SS）将酸性多糖褐藻胶纳入细胞质。我们通过在钙结合位点引入突变来研究 AlgQ2 中 EF 手样结构域中结合的钙的功能。AlgQ2 突变体（D179A/E180A）的 X 射线晶体学表明，没有钙结合，EF 手样结构域的显著无序。与野生型 AlgQ2 不同的是，尽管不饱和褐藻胶低聚糖通过形成底物/蛋白复合物稳定了突变体，但在 A1 生长温度下，突变体相当不稳定。在脂质体中重建的 AlgM1M2SS 的 ATP 酶和褐藻胶转运测定中，野生型和突变型 AlgQ2 在存在不饱和褐藻胶四糖的情况下诱导 AlgM1M2SS ATP 酶活性。这些结果表明，EF 手样结构域结合的钙稳定未结合底物的 AlgQ2，但不需要结合底物的 AlgQ2 和 AlgM1M2SS 的复合。
