Structural Analysis of the Catalytic Mechanism and Substrate Specificity of Anabaena Alkaline Invertase InvA Reveals a Novel Glucosidase

作者：Jin Xie, 进 谢, Kun Cai, 坤 蔡, Hai‐Xi Hu, 海汐 胡, Yong‐Liang Jiang, 永亮 江, Feng Yang, 丰 杨, Pengfei Hu, 鹏飞 胡, Dongdong Cao, 冬冬 曹, Weifang Li, 卫芳 李, Yuxing Chen, 宇星 陈, Cong‐Zhao Zhou, 丛照 周 · 发表于：Journal of Biological Chemistry · 年份：2016 · DOI：10.1074/jbc.m116.759290 · 被引用次数：37 · 研究领域：Microbial Metabolites in Food Biotechnology、Enzyme Structure and Function、Enzyme Production and Characterization

Invertases catalyze the hydrolysis of sucrose to glucose and fructose, thereby playing a key role in primary metabolism and plant development. According to the optimum pH, invertases are classified into acid invertases (Ac-Invs) and alkaline/neutral invertases (A/N-Invs), which share no sequence homology. Compared with Ac-Invs that have been extensively studied, the structure and catalytic mechanism of A/N-Invs remain unknown. Here we report the crystal structures of Anabaena alkaline invertase InvA, which was proposed to be the ancestor of modern plant A/N-Invs. These structures are the first in the GH100 family. InvA exists as a hexamer in both crystal and solution. Each subunit consists of an (α/α) 6 barrel core structure in addition to an insertion of three helices. A couple of structures in complex with the substrate or products enabled us to assign the subsites −1 and +1 specifically binding glucose and fructose, respectively. Structural comparison combined with enzymatic assays indicated that Asp-188 and Glu-414 are putative catalytic residues. Further analysis of the substrate binding pocket demonstrated that InvA possesses a stringent substrate specificity toward the α1,2-glycosidic bond of sucrose. Together, we suggest that InvA and homologs represent a novel family of glucosidases.