Structures of PspAG97A α-glucoside hydrolase reveal a novel mechanism for chloride induced activation
Structures of PspAG97A α-glucoside hydrolase reveal a novel mechanism for chloride induced activation
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PspAG97A α-葡萄糖苷水解酶的结构揭示了氯诱导激活的新机制
DOI：
10.1016/j.jsb.2016.09.009
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发表时间：
2016-12-01
影响因子：
3
通讯作者：
Xiao, Yazhong
中科院分区：
文献类型：
--
作者：
He, Chao;Li, Jing;Xiao, Yazhong
本文首次报道了从假交替单胞菌K8中分离到的一种分泌型α-葡萄糖苷水解酶PspAG 97 A的晶体结构，该酶属于糖苷水解酶家族97，具有嗜盐性。PspAG 97 A缺乏酸性表面，这被认为是在高盐度下蛋白质稳定性所必需的。有趣的是，PspAG 97 A不寻常地包含由Arg 171的胍基和Tyr 172和Glu 173的主链酰胺基在活性位点配位的氯离子。与阿卡波糖和潘糖复合的PspAG 97 A的结构表明，亚位点+1的残基Glu 173、Arg 171和Asn 170决定了酶对α-1，6-糖苷键的底物特异性。在R171 K变体和酶动力学实验中观察到的结构改变集中于氯离子辅助活化，表明活性位点氯离子用于适当定向Glul 73、Arg 171和Asn 170以促进底物识别。此外，氯化物有助于Glul 73与保守的钙离子结合，并在适当定位碱催化剂Glul 456中起重要作用。总之，我们的研究结果提供了有价值的洞察蛋白质嗜盐性的结构基础。(C)2016 Elsevier Inc. All rights reserved.
Here we report the first crystal structure of a secretory cc-glucoside hydrolase isolated from Pseudoalteromonas sp. K8, PspAG97A, which belongs to glycoside hydrolase family 97 and exhibits halophilic property. PspAG97A lacks an acidic surface, that is considered essential for protein stability at high salinity. Interestingly, PspAG97A unusually contains a chloride ion coordinated by the guanidinium group of Arg171 and the main chain amide groups of Tyr172 and Glu173 at the active site. The structures of PspAG97A complexed with acarbose and panose demonstrate that residues Glu173, Arg171 and Asn170 for subsite +1 decide the substrate specificity of the enzyme for the alpha-1,6-glucosidic linkage. Structural alterations observed in the R171K variant and enzyme kinetic experiments focusing on chloride assisted activation suggest that the active site chloride serves to properly orient Glul 73, Arg171 and Asn170 to facilitate substrate recognition. Furthermore, the chloride assists the binding of Glul 73 to the conserved calcium ion and plays an essential role in properly positioning the base catalyst Glu456. In sum, our results provide valuable insight into the structural basis of protein halophilicity. (C) 2016 Elsevier Inc. All rights reserved.