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首页> 外文期刊>Journal of Molecular Biology >Crystal structures of the bacterial solute receptor AcbH displaying an exclusive substrate preference for beta-D-galactopyranose.
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Crystal structures of the bacterial solute receptor AcbH displaying an exclusive substrate preference for beta-D-galactopyranose.

机译：细菌溶质受体AcbH的晶体结构显示出对β-D-吡喃半乳糖的专有底物偏好。

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摘要

Solute receptors (binding proteins) are indispensable components of canonical ATP-binding cassette importers in prokaryotes. Here, we report on the characterization and crystal structures in the closed and open conformations of AcbH, the solute receptor of the putative carbohydrate transporter AcbFG which is encoded in the acarbose (acarviosyl-1,4-maltose) biosynthetic gene cluster from Actinoplanes sp. SE50/110. Binding assays identified AcbH as a high-affinity monosaccharide-binding protein with a dissociation constant (K(d)) for beta-d-galactopyranose of 9.8+/-1.0 nM. Neither galactose-containing di- and trisaccharides, such as lactose and raffinose, nor monosaccharides including d-galacturonic acid, l-arabinose, d-xylose and l-rhamnose competed with [(1)(4)C]galactose for binding to AcbH. Moreover, AcbH does not bind d-glucose, which is a common property of all but one d-galactose-binding proteins characterized to date. Strikingly, determination of the X-ray structure revealed that AcbH is structurally homologous to maltose-binding proteins rather than to glucose-binding proteins. Two helices are inserted in the substrate-binding pocket, which reduces the cavity size and allows the exclusive binding of monosaccharides, specifically beta-d-galactopyranose, in the (4)C(1) conformation. Site-directed mutagenesis of three residues from the binding pocket (Arg82, Asp361 and Arg362) that interact with the axially oriented O4-H hydroxyl of the bound galactopyranose and subsequent functional analysis indicated that these residues are crucial for galactose binding. To our knowledge, this is the first report of the tertiary structure of a solute receptor with exclusive affinity for beta-d-galactopyranose. The putative role of a galactose import system in the context of acarbose metabolism in Actinoplanes sp. is discussed.
机译：溶质受体（结合蛋白）是原核生物中经典的ATP结合盒导入物必不可少的组成部分。在这里，我们报告AcbH的封闭和开放构象的特征和晶体结构，AcbH是推定的碳水化合物转运蛋白AcbFG的溶质受体，编码在Actinoplanes sp。的阿卡波糖（acarviosyl-1,4-麦芽糖）生物合成基因簇中。 SE50 / 110。结合测定法确定AcbH为高亲和力单糖结合蛋白，β-d-吡喃半乳糖的解离常数（K（d））为9.8 +/- 1.0 nM。含半乳糖的二糖和三糖，例如乳糖和棉子糖，或包括d-半乳糖醛酸，l-阿拉伯糖，d-木糖和l-鼠李糖在内的单糖均未与[（1）（4）C]半乳糖竞争与AcbH的结合。 。此外，AcbH不结合d-葡萄糖，这是迄今为止已表征的除一种d-半乳糖结合蛋白以外所有蛋白的共同特性。惊人地，对X射线结构的测定表明，AcbH在结构上与麦芽糖结合蛋白而不是与葡萄糖结合蛋白同源。两个螺旋插入底物结合袋中，这减小了空腔的大小，并允许单糖（特别是β-d-吡喃半乳糖）以（4）C（1）构型排他地结合。从结合袋中的三个残基（Arg82，Asp361和Arg362）与结合的半乳糖吡喃糖的轴向O4-H羟基相互作用的定点诱变，随后的功能分析表明，这些残基对于半乳糖结合至关重要。据我们所知，这是对β-d-吡喃半乳糖具有唯一亲和力的溶质受体三级结构的首次报道。在放线猕猴桃中的阿卡波糖代谢中，半乳糖输入系统的推定作用。讨论。

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