Shanghai Key Laboratory of Agricultural Genetics and Breeding, Biotechnology Research Institute, Shanghai Academy of Agricultural Sciences, Shanghai, People's Republic of China.
PLoS One. 2012;7(8):e39579. doi: 10.1371/journal.pone.0039579. Epub 2012 Aug 3.
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.
5-烯醇丙酮酰莽草酸-3-磷酸合酶（EPSPS；EC 2.5.1.19）是植物、真菌和微生物中芳香族氨基酸和分支酸衍生次生代谢物生物合成途径中的关键酶，也是广谱除草剂草甘膦的靶标。天然草甘膦抗性通常被认为是在微生物中强烈选择压力条件下发生的。从受草甘膦污染的葡萄园葡萄根际中分离到一种拮抗葡萄冠瘿病菌的拮抗菌 Rhahnella aquatilis 菌株 GR20。通过互补大肠杆菌营养缺陷型 aroA 突变体，从分离的细菌中鉴定出一种新型编码 EPSPS 的基因。该 EPSPS 命名为 AroA（R. aquatilis），从大肠杆菌中表达和纯化，并测定了关键动力学值。全长酶对草甘膦的耐受性高于大肠杆菌 EPSPS（AroA（E. coli）），同时对底物磷酸烯醇丙酮酸保持高亲和力。与 AroA（E. coli）相比，转 AroA（R. aquatilis）植物对浓度为 5mM 的草甘膦也表现出更高的抗性。为了探究导致对草甘膦耐受性增加的位点，用大肠杆菌 EPSPS 的 c 链和亚结构域 5 的 f 链（Thr38Lys、Arg40Val、Arg222Gln、Ser224Val、Ile225Val 和 Gln226Lys）取代突变的 R. aquatilis EPSPS 酶，产生突变体。与野生型 R. aquatilis EPSPS 相比，突变酶对草甘膦的敏感性更高，而对其第一底物莽草酸-3-磷酸（S3P）和磷酸烯醇丙酮酸（PEP）的亲和力变化不大。亚结构域 5 上残基对草甘膦抗性的影响更为明显。
