School of Food Science and Technology, Jiangnan University, Wuxi, 214122, China.
USDA-ARS West Research Center, 800 Buchanan St, Albany, CA, 74710, USA.
Appl Biochem Biotechnol. 2020 Mar;190(3):1010-1022. doi: 10.1007/s12010-019-03150-7. Epub 2019 Oct 25.
By sequence comparison, the majority of 1,4-α-glucan-branching enzymes (GBEs) consist of an N-terminal carbohydrate-binding domain, a TIM-barrel catalytic domain, and a C-terminal all-beta domain. Among these structures, the GBE from Geobacillus thermoglucosidans STB02 uniquely has a highly charged 26-amino-acid C-terminal extension, whose functional roles are the least understood. In this research, the functional significance of the C-terminal domain in GBE from G. thermoglucosidans STB02 and its extension were assessed using a C-terminal deletion analysis. Mutants lacking of more than 7 residues of the C-terminal all-beta domain could not be detected in lysates of their Escherichia coli expression strains, suggesting that an intact all-beta domain is required for structural stability. In contrast, truncation of the C-terminal extension resulted in greater stability and solubility than the wild type, as well as a lower sensitivity to the presence of added metal ions. Comparison of this mutant with the wild type suggests that the interaction of metal ions with the C-terminal extension influences performance of this enzyme.
通过序列比较，大多数 1,4-α-葡聚糖分支酶（GBE）由一个 N 端碳水化合物结合结构域、一个 TIM 桶催化结构域和一个 C 端全β结构域组成。在这些结构中，来自嗜热解糖梭菌 STB02 的 GBE 具有独特的高电荷 26 个氨基酸 C 端延伸，其功能作用了解最少。在这项研究中，使用 C 端缺失分析评估了来自嗜热解糖梭菌 STB02 的 GBE 中 C 端结构域及其延伸的功能意义。在其大肠杆菌表达菌株的裂解物中无法检测到缺失超过 7 个 C 端全β结构域残基的突变体，这表明完整的全β结构域对于结构稳定性是必需的。相比之下，C 端延伸的截断导致比野生型更高的稳定性和溶解度，以及对添加金属离子的敏感性降低。与野生型的比较表明，金属离子与 C 端延伸的相互作用影响了该酶的性能。
