title: Improvement of glyphosate resistance through concurrent mutations in three amino acids of the Ochrobactrum 5-enopyruvylshikimate-3-phosphate synthase.
abstract: A mutant of 5-enopyruvylshikimate-3-phosphate synthase from Ochrobactrum anthropi was identified after four rounds of DNA shuffling and screening. Its ability to restore the growth of the mutant ER2799 cell on an M9 minimal medium containing 300 mM glyphosate led to its identification. The mutant had mutations in seven amino acids: E145G, N163H, N267S, P318R, M377V, M425T, and P438L. Among these mutations, N267S, P318R, and M425T have never been previously reported as important residues for glyphosate resistance. However, in the present study they were found by site-directed mutagenesis to collectively contribute to the improvement of glyphosate tolerance. Kinetic analyses of these three mutants demonstrated that the effectiveness of these three individual amino acid alterations on glyphosate tolerance was in the order P318R > M425T > N267S. The results of the kinetic analyses combined with a three-dimensional structure modeling of the location of P318R and M425T demonstrate that the lower hemisphere's upper surface is possibly another important region for glyphosate resistance. Furthermore, the transgenic Arabidopsis was obtained to confirm the potential of the mutant in developing glyphosate-resistant crops.
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, 2901 Beidi Rd., Shanghai, People's Republic of China.
authors.forename: Yong-Sheng
authors.initials: YS
authors.lastname: Tian
authors.forename: Jing
authors.initials: J
authors.lastname: Xu
authors.forename: Ai-Sheng
authors.initials: AS
authors.lastname: Xiong
authors.forename: Wei
authors.initials: W
authors.lastname: Zhao
authors.forename: Xiao-Yan
authors.initials: XY
authors.lastname: Fu
authors.forename: Ri-He
authors.initials: RH
authors.lastname: Peng
authors.forename: Quan-Hong
authors.initials: QH
authors.lastname: Yao
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D019943:Amino Acid Substitution; D000900:Anti-Bacterial Agents / Q000378:metabolism*; D017360:Arabidopsis / Q000187:drug effects / Q000235:genetics; D032621:DNA Shuffling; D024881:Drug Resistance, Bacterial; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D006540:Herbicides / Q000494:pharmacology*; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D020621:Ochrobactrum anthropi / Q000187:drug effects* / Q000201:enzymology / Q000235:genetics; D030821:Plants, Genetically Modified / Q000187:drug effects / Q000235:genetics; D011487:Protein Conformation; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D000900:Anti-Bacterial Agents; D005998:Glycine; D006540:Herbicides; D050505:Mutant Proteins; D000097797:Glyphosate