title: Alanine 310 is important for the activity of 1,4-α-glucan branching enzyme from Geobacillus thermoglucosidans STB02.
abstract: 1,4-α-Glucan branching enzyme (GBE) catalyzes the formation of α-1,6 branch points in starch or glycogen by hydrolyzing α-1,4-glucosidic linkages and then synthesizing α-1,6-glucosidic linkages. In the GBE from Geobacillus thermoglucosidans STB02, alanine 310 (Ala310) is located in conserved region II. An analysis of the amino acid sequence shows that Ala310 is highly conserved in the prokaryotic GBE subfamily. Site-directed mutagenesis was used to determine the function of Ala310 in GBE. Replacement of Ala310 with glycine, aspartate, asparagine, isoleucine, glutamate, or glutamine resulted in mutant enzymes with less than 10% to 25% of wild-type activity when amylopectin or amylose was used as substrate. Studies using high-performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD) showed that A310G mutant had no effect on the transfer pattern, but the branching activity had been repressed to a large extent. Kinetic analysis also showed that mutations of Ala310 had an effect on the KMvalue that changed the preferred substrate from amylopectin to amylose. These results show that Ala310 is important for the catalytic activity of the GBE from G. thermoglucosidans STB02.
authors.affiliation: School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors.forename: Yiting
authors.initials: Y
authors.lastname: Liu
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China.
authors.forename: Caiming
authors.initials: C
authors.lastname: Li
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China. Electronic address: zhengbiaogu@jiangnan.edu.cn.
authors.forename: Zhengbiao
authors.initials: Z
authors.lastname: Gu
authors.affiliation: School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors.forename: Chenhao
authors.initials: C
authors.lastname: Xin
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China.
authors.forename: Li
authors.initials: L
authors.lastname: Cheng
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Hong
authors.affiliation: State Key Laboratory of Food Science and Technology, Jiangnan University, Wuxi 214122, China; School of Food Science and Technology, Jiangnan University, Wuxi 214122, China; Synergetic Innovation Center of Food Safety and Nutrition, Jiangnan University, Wuxi, Jiangsu 214122, China. Electronic address: zfli@jiangnan.edu.cn.
authors.forename: Zhaofeng
authors.initials: Z
authors.lastname: Li
mesh_terms: D015061:1,4-alpha-Glucan Branching Enzyme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D000409:Alanine; D055162:Biocatalysis; D056506:Geobacillus / Q000201:enzymology*; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D011487:Protein Conformation; D017421:Sequence Analysis
chemical_list: D015061:1,4-alpha-Glucan Branching Enzyme; D000409:Alanine
keywords: 1,4-α-Glucan branching enzyme; Alanine310; Enzymatic activity; Site-directed mutagenesis; Substrate preference