title: Structural Analysis of the Catalytic Mechanism and Substrate Specificity of Anabaena Alkaline Invertase InvA Reveals a Novel Glucosidase.
abstract: 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)6barrel 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.
authors: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China.
authors: Jin
authors: J
authors: Xie
authors: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China.
authors: Kun
authors: K
authors: Cai
authors: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China.
authors: Hai-Xi
authors: HX
authors: Hu
authors: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China.
authors: Yong-Liang
authors: YL
authors: Jiang
authors: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China.
authors: Feng
authors: F
authors: Yang
authors: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China.
authors: Peng-Fei
authors: PF
authors: Hu
authors: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China.
authors: Dong-Dong
authors: DD
authors: Cao
authors: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China.
authors: Wei-Fang
authors: WF
authors: Li
authors: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China cyxing@ustc.edu.cn.
authors: Yuxing
authors: Y
authors: Chen
authors: From the Hefei National Laboratory for Physical Sciences at the Microscale and School of Life Sciences, University of Science and Technology of China, Hefei Anhui 230027, China zcz@ustc.edu.cn.
authors: Cong-Zhao
authors: CZ
authors: Zhou
mesh_terms: D017033:Anabaena / Q000201:enzymology* / Q000235:genetics; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D018360:Crystallography, X-Ray; D005632:Fructose / Q000737:chemistry / Q000378:metabolism; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D000072417:Protein Domains; D013395:Sucrose / Q000737:chemistry / Q000378:metabolism; D043324:beta-Fructofuranosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D005632:Fructose; D013395:Sucrose; D043324:beta-Fructofuranosidase; D005947:Glucose
keywords: GH100; alkaline/neutral invertases; crystal structure; cyanobacteria; enzyme catalysis; glucosidase; glycoside hydrolase; substrate specificity; sucrose metabolism