title: Improvement of glycine oxidase by DNA shuffling, and site-saturation mutagenesis of F247 residue.
abstract: Glyphosate is a broad spectrum herbicide widely used throughout the world, and it could be degraded by glycine oxidase (GO) through CN bond cleavage. For a better understanding of the structure-function relationship and improving the activity of B3S1 (GO from Bacillus cereus), DNA shuffling was performed. A mutant B4S7 (The Km, Vmax, kcat and kcat/Km values on glyphosate were 0.1 mM, 0.002401 mM min(-1), 3.62 min(-1) and 36.2 mM(-1) min(-1), respectively. The four parameters on glycine were 50.34 mM, 0.001983 mM min(-1), 2.18 min(-1) and 0.04 mM(-1) min(-1), respectively) was obtained from 10,000 clones, which presented a 3.9-fold increase of the specificity constant (the kcat/Km ratio between glyphosate and glycine) compared with B3S1. Especially, the Km value of B4S7 to glyphosate was much less than those of the reported GO. Structure modeling and molecular docking indicated that the novel mutation point F247S was close to the active site of the enzyme. To identify the role of the site, the remaining 19 amino acids were introduced into the site by site-saturation mutagenesis. The result showed that compared with B3S1, the specificity constant of mutant F247S and F247R increased 0.64-fold and 1.04-fold, respectively. While the specificity constant of mutant F247E decreased 2.01-fold. Therefore, the site 247 plays a crucial role in regulating the substrate specificity. This study provides new information on the structure-function relationship of glycine oxidase and the development of glyphosate-tolerant crops.
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Pei
authors.initials: P
authors.lastname: Yao
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Lin
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Gaobing
authors.initials: G
authors.lastname: Wu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Yulin
authors.initials: Y
authors.lastname: Lu
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Tao
authors.initials: T
authors.lastname: Zhan
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China.
authors.forename: Ashok
authors.initials: A
authors.lastname: Kumar
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China; Key Laboratory of Protection and Utilization of Biological Resources in Tarim Basin of Xinjiang Production and Construction Crops, College of Life Science, Tarim University, Alar 843300, China. Electronic address: zhang63lyly@sina.com.
authors.forename: Lili
authors.initials: L
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Agricultural Microbiology, College of Life Science and Technology, Huazhong Agricultural University, Wuhan 430070, China. Electronic address: lzd@mail.hzau.edu.cn.
authors.forename: Ziduo
authors.initials: Z
authors.lastname: Liu
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D019943:Amino Acid Substitution / Q000235:genetics; D001409:Bacillus cereus / Q000201:enzymology*; D032621:DNA Shuffling; D005998:Glycine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006540:Herbicides / Q000737:chemistry*; D007700:Kinetics; D062105:Molecular Docking Simulation; D016297:Mutagenesis, Site-Directed; D030821:Plants, Genetically Modified / Q000201:enzymology; D013379:Substrate Specificity; D000097797:Glyphosate
chemical_list: D000594:Amino Acid Oxidoreductases; D005998:Glycine; D006540:Herbicides; D000097797:Glyphosate; C115925:glycine oxidase
keywords: DNA shuffling; Glycine oxidase; Glyphosate; Site-saturation mutagenesis; Substrate specificity