Laboratory of Basic and Applied Molecular Biotechnology, Graduate School of Agriculture, Kyoto University, Uji, Kyoto 611-0011, Japan.
Biochemistry. 2012 May 1;51(17):3622-33. doi: 10.1021/bi300194f. Epub 2012 Apr 23.
Alginate is a heteropolysaccharide that consists of β-D-mannuronate (M) and α-L-guluronate (G). The Gram-negative bacterium Sphingomonas sp. A1 directly incorporates alginate into the cytoplasm through the periplasmic solute-binding protein (AlgQ1 and AlgQ2)-dependent ABC transporter (AlgM1-AlgM2/AlgS-AlgS). Two binding proteins with at least four subsites strongly recognize the nonreducing terminal residue of alginate at subsite 1. Here, we show the broad substrate preference of strain A1 solute-binding proteins for M and G present in alginate and demonstrate the structural determinants in binding proteins for heteropolysaccharide recognition through X-ray crystallography of four AlgQ1 structures in complex with saturated and unsaturated alginate oligosaccharides. Alginates with different M/G ratios were assimilated by strain A1 cells and bound to AlgQ1 and AlgQ2. Crystal structures of oligosaccharide-bound forms revealed that in addition to interaction between AlgQ1 and unsaturated oligosaccharides, the binding protein binds through hydrogen bonds to the C4 hydroxyl group of the saturated nonreducing terminal residue at subsite 1. The M residue of saturated oligosaccharides is predominantly accommodated at subsite 1 because of the strict binding of Ser-273 to the carboxyl group of the residue. In unsaturated trisaccharide (ΔGGG or ΔMMM)-bound AlgQ1, the protein interacts appropriately with substrate hydroxyl groups at subsites 2 and 3 to accommodate M or G, while substrate carboxyl groups are strictly recognized by the specific residues Tyr-129 at subsite 2 and Lys-22 at subsite 3. Because of this substrate recognition mechanism, strain A1 solute-binding proteins can bind heteropolysaccharide alginate with different M/G ratios.
藻酸盐是一种杂多糖，由β-D-甘露糖醛酸（M）和α-L-古洛糖醛酸（G）组成。革兰氏阴性菌鞘氨醇单胞菌 A1 通过周质溶质结合蛋白（AlgQ1 和 AlgQ2）依赖性 ABC 转运体（AlgM1-AlgM2/AlgS-AlgS）直接将藻酸盐纳入细胞质。至少有四个亚基的两个结合蛋白在亚基 1 处强烈识别藻酸盐的非还原末端残基。在这里，我们展示了 A1 溶质结合蛋白对藻酸盐中存在的 M 和 G 的广泛底物偏好，并通过与饱和和不饱和藻酸盐寡糖复合物的四个 AlgQ1 结构的 X 射线晶体学显示结合蛋白中结构决定因素用于识别杂多糖。具有不同 M/G 比的藻酸盐被 A1 细胞同化，并与 AlgQ1 和 AlgQ2 结合。寡糖结合形式的晶体结构表明，除了 AlgQ1 与不饱和寡糖之间的相互作用外，结合蛋白还通过氢键与亚基 1 处的饱和非还原末端残基的 C4 羟基结合。由于 Ser-273 与残基的羧基严格结合，饱和寡糖的 M 残基主要容纳在亚基 1 中。在不饱和三糖（ΔGGG 或 ΔMMM）结合的 AlgQ1 中，蛋白质在亚基 2 和 3 处与底物羟基适当相互作用以容纳 M 或 G，而底物羧基基团则由亚基 2 处的特异残基 Tyr-129 和亚基 3 处的 Lys-22 严格识别。由于这种底物识别机制，A1 溶质结合蛋白可以结合具有不同 M/G 比的杂多糖藻酸盐。
