Department of Manufacturing Pharmacy, College of Pharmacy and Research Institute for Drug Development, Pusan National University, Geumjeong-gu, Busan 609-735, Republic of Korea.
Biochem Biophys Res Commun. 2011 Dec 9;416(1-2):92-8. doi: 10.1016/j.bbrc.2011.11.002. Epub 2011 Nov 10.
Glycoside hydrolase family 4 (GH4) represents an unusual group of glucosidases with a requirement for NAD(+), Mn(2+), and reducing conditions. We found a putative α-glucosidase belonging to GH4 in hyperthermophilic Gram-negative bacterium Thermotoga neapolitana. In this study, we recombinantly expressed the putative α-glycosidase from T. neapolitana, and determined the crystal structure of the protein at a resolution of 2.0Å in the presence of Mn(2+) but in the absence of NAD(+). The structure showed the dimeric assembly and the Mn(2+) coordination that other GH4 enzymes share. In comparison, we observed structural changes in T. neapolitana α-glucosidase by the binding of NAD(+), which also increased the thermostability. Numerous arginine-mediated salt-bridges were observed in the structure, and we confirmed that the salt bridges correlated with the thermostability of the proteins. Disruption of the salt bridge that linked N-terminal and C-terminal parts at the surface dramatically decreased the thermostability. A mutation that changed the internal salt bridge to a hydrogen bond also decreased the thermostability of the protein. This study will help us to understand the function of the putative glucosidase and the structural features that affect the thermostability of the protein.
糖苷水解酶家族 4（GH4）代表了一类具有独特性质的葡萄糖苷酶，它们需要 NAD(+)、Mn(2+) 和还原条件。我们在嗜热革兰氏阴性菌 Thermotoga neapolitana 中发现了一种假定的属于 GH4 的α-葡萄糖苷酶。在本研究中，我们重组表达了来自 T. neapolitana 的假定α-糖苷酶，并在存在 Mn(2+)但不存在 NAD(+)的情况下，以 2.0Å 的分辨率确定了该蛋白的晶体结构。该结构显示了其他 GH4 酶所共有的二聚体组装和 Mn(2+)配位。相比之下，我们观察到 NAD(+)结合引起了 T. neapolitana α-葡萄糖苷酶的结构变化，这也增加了其热稳定性。在结构中观察到许多精氨酸介导的盐桥，我们证实这些盐桥与蛋白质的热稳定性相关。在表面破坏连接 N 端和 C 端部分的盐桥会显著降低热稳定性。将内部盐桥改变为氢键的突变也降低了蛋白质的热稳定性。本研究将有助于我们理解假定葡萄糖苷酶的功能以及影响蛋白质热稳定性的结构特征。
