title: A novel 5-enolpyruvylshikimate-3-phosphate synthase from Rahnella aquatilis with significantly reduced glyphosate sensitivity.
abstract: The 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS; EC 2.5.1.19) is a key enzyme in the shikimate pathway for the production of aromatic amino acids and chorismate-derived secondary metabolites in plants, fungi, and microorganisms. It is also the target of the broad-spectrum herbicide glyphosate. Natural glyphosate resistance is generally thought to occur within microorganisms in a strong selective pressure condition. Rahnella aquatilis strain GR20, an antagonist against pathogenic agrobacterial strains of grape crown gall, was isolated from the rhizosphere of grape in glyphosate-contaminated vineyards. A novel gene encoding EPSPS was identified from the isolated bacterium by complementation of an Escherichia coli auxotrophic aroA mutant. The EPSPS, named AroA(R. aquatilis), was expressed and purified from E. coli, and key kinetic values were determined. The full-length enzyme exhibited higher tolerance to glyphosate than the E. coli EPSPS (AroA(E. coli)), while retaining high affinity for the substrate phosphoenolpyruvate. Transgenic plants of AroA(R. aquatilis) were also observed to be more resistant to glyphosate at a concentration of 5 mM than that of AroA(E. coli). To probe the sites contributing to increased tolerance to glyphosate, mutant R. aquatilis EPSPS enzymes were produced with the c-strand of subdomain 3 and the f-strand of subdomain 5 (Thr38Lys, Arg40Val, Arg222Gln, Ser224Val, Ile225Val, and Gln226Lys) substituted by the corresponding region of the E. coli EPSPS. The mutant enzyme exhibited greater sensitivity to glyphosate than the wild type R. aquatilis EPSPS with little change of affinity for its first substrate, shikimate-3-phosphate (S3P) and phosphoenolpyruvate (PEP). The effect of the residues on subdomain 5 on glyphosate resistance was more obvious.
authors.affiliation: Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, People's Republic of China.
authors.forename: Ri-He
authors.initials: RH
authors.lastname: Peng
authors.forename: Yong-Sheng
authors.initials: YS
authors.lastname: Tian
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: Hong-Juan
authors.initials: HJ
authors.lastname: Han
authors.forename: Chen
authors.initials: C
authors.lastname: Chen
authors.forename: Xiao-Fen
authors.initials: XF
authors.lastname: Jin
authors.forename: Quan-Hong
authors.initials: QH
authors.lastname: Yao
mesh_terms: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase / Q000235:genetics / Q000378:metabolism; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D001483:Base Sequence; D024881:Drug Resistance, Bacterial / Q000502:physiology*; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D005998:Glycine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008969:Molecular Sequence Data; D020125:Mutation, Missense; D010935:Plant Diseases / Q000382:microbiology; D017434:Protein Structure, Tertiary; D020638:Rahnella / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity; D027843:Vitis / Q000382:microbiology; D000097797:Glyphosate
chemical_list: D051229:3-Phosphoshikimate 1-Carboxyvinyltransferase; D001426:Bacterial Proteins; D004791:Enzyme Inhibitors; D005998:Glycine; D000097797:Glyphosate