Institut für Chemie und Biochemie/Kristallographie, Freie Universität Berlin, Berlin, Germany.
J Mol Biol. 2010 Apr 2;397(3):709-23. doi: 10.1016/j.jmb.2010.01.054. Epub 2010 Feb 2.
GacH is the solute binding protein (receptor) of the putative oligosaccharide ATP-binding cassette transporter GacFG, encoded in the acarbose biosynthetic gene cluster (gac) from Streptomyces glaucescens GLA.O. In the context of the proposed function of acarbose (acarviosyl-1,4-maltose) as a 'carbophor,' the transporter, in complex with a yet to be identified ATPase subunit, is supposed to mediate the uptake of longer acarbose homologs and acarbose for recycling purposes. Binding assays using isothermal titration calorimetry identified GacH as a maltose/maltodextrin-binding protein with a low affinity for acarbose but with considerable binding activity for its homolog, component 5C (acarviosyl-1,4-maltose-1,4-glucose-1,1-glucose). In contrast, the maltose-binding protein of Salmonella typhimurium (MalE) displays high-affinity acarbose binding. We determined the crystal structures of GacH in complex with acarbose, component 5C, and maltotetraose, as well as in unliganded form. As found for other solute receptors, the polypeptide chain of GacH is folded into two distinct domains (lobes) connected by a hinge, with the interface between the lobes forming the substrate-binding pocket. GacH does not specifically bind the acarviosyl group, but displays specificity for binding of the maltose moiety in the inner part of its binding pocket. The crystal structure of acarbose-loaded MalE showed that two glucose units of acarbose are bound at the same region and position as maltose. A comparative analysis revealed that in GacH, acarbose is buried deeper into the binding pocket than in MalE by exactly one glucose ring shift, resulting in a total of 18 hydrogen-bond interactions versus 21 hydrogen-bond interactions for MalE(acarbose). Since the substrate specificity of ATP-binding cassette import systems is determined by the cognate binding protein, our results provide the first biochemical and structural evidence for the proposed role of GacHFG in acarbose metabolism.
GacH 是假定寡糖 ATP 结合盒转运蛋白 GacFG 的溶质结合蛋白（受体），该蛋白编码于链霉菌（Streptomyces glaucescens GLA.O）的阿卡波糖生物合成基因簇（gac）中。在阿卡波糖（阿卡波糖基-1,4-麦芽糖）作为“carbophor”的拟议功能背景下，该转运蛋白与尚未鉴定的 ATP 酶亚基形成复合物，据推测介导了更长的阿卡波糖类似物和阿卡波糖的摄取，用于回收利用。使用等温滴定量热法进行的结合测定将 GacH 鉴定为低亲和力结合阿卡波糖但对其类似物组分 5C（阿卡波糖基-1,4-麦芽糖-1,4-葡萄糖-1,1-葡萄糖）具有相当结合活性的麦芽糖/麦芽糖糊精结合蛋白。相比之下，鼠伤寒沙门氏菌（Salmonella typhimurium）的麦芽糖结合蛋白（MalE）显示出对阿卡波糖的高亲和力结合。我们确定了 GacH 与阿卡波糖、组分 5C 和麦芽四糖复合物以及无配体形式的晶体结构。与其他溶质受体一样，GacH 的多肽链折叠成两个通过铰链连接的不同结构域（叶），叶之间的界面形成底物结合口袋。GacH 不特异性结合阿卡波糖基，但显示出对其结合口袋内部的麦芽糖部分的特异性结合。负载阿卡波糖的 MalE 的晶体结构表明，阿卡波糖的两个葡萄糖单位与麦芽糖相同的区域和位置结合。比较分析表明，在 GacH 中，由于一个葡萄糖环的移位，阿卡波糖比 MalE 更深地埋入结合口袋中，导致总共 18 个氢键相互作用，而 MalE（阿卡波糖）的氢键相互作用为 21 个。由于 ATP 结合盒导入系统的底物特异性由同源结合蛋白决定，我们的结果为 GacHFG 在阿卡波糖代谢中的拟议作用提供了第一个生化和结构证据。
