Structural basis of glyphosate tolerance resulting from mutations of Pro101 in Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase
Structural basis of glyphosate tolerance resulting from mutations of Pro101 in Escherichia coli 5-enolpyruvylshikimate-3-phosphate synthase
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DOI：
10.1074/jbc.m705624200
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发表时间：
2007-11-09
影响因子：
4.8
通讯作者：
Schoenbrunn, Ernst
中科院分区：
文献类型：
--
作者：
Healy-Fried, Martha L.;Funke, Todd;Schoenbrunn, Ernst
草甘膦是世界上使用最多的除草剂，它取得了巨大的成功，因为它能够以最小的动物和环境毒性有效地控制杂草。草甘膦的分子靶标是5-烯醇式莽草酸-3-磷酸合酶（EPSPS），它催化植物和微生物中莽草酸途径的第六步。EPSPS的草甘膦耐受变体构成了基因工程除草剂耐受作物的基础。EPSPS中Pro（101）的单位点突变（根据大肠杆菌的酶编号）与草甘膦抗性杂草有关，但该残基并不直接参与草甘膦结合，在缺乏进一步动力学和结构表征的情况下，这种现象的基础仍然不清楚。为了探讨该位点突变的影响，E.用甘氨酸、丙氨酸、丝氨酸或亮氨酸取代Pro（101）产生大肠杆菌EPSPS酶。通过稳态动力学以及底物二元物和底物的晶体结构来分析这些突变酶。测定P101 S和P101 LEPSPS的草甘膦三元复合物的分辨率在1.5- 1.6埃之间。看起来比亮氨酸小的残基可以取代Pro（101）而不降低催化效率。该位点的任何突变都会导致草甘膦结合位点的结构变化，使Thr（97）和Gly（96）向抑制剂分子转移。我们的结论是，观察到草甘膦的抑制效力降低是这些突变引起的长期结构变化的结果。我们的研究结果的发展和传播的抗草甘膦杂草的影响进行了讨论。
Glyphosate, the world's most used herbicide, is a massive success because it enables efficient weed control with minimal animal and environmental toxicity. The molecular target of glyphosate is 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS), which catalyzes the sixth step of the shikimate pathway in plants and microorganisms. Glyphosate-tolerant variants of EPSPS constitute the basis of genetically engineered herbicide-tolerant crops. A single-site mutation of Pro(101) in EPSPS (numbering according to the enzyme from Escherichia coli) has been implicated in glyphosate-resistant weeds, but this residue is not directly involved in glyphosate binding, and the basis for this phenomenon has remained unclear in the absence of further kinetic and structural characterization. To probe the effects of mutations at this site, E. coli EPSPS enzymes were produced with glycine, alanine, serine, or leucine substituted for Pro(101). These mutant enzymes were analyzed by steady-state kinetics, and the crystal structures of the substrate binary and substrate . glyphosate ternary complexes of P101S and P101L EPSPS were determined to between 1.5- and 1.6-angstrom resolution. It appears that residues smaller than leucine may be substituted for Pro(101) without decreasing catalytic efficiency. Any mutation at this site results in a structural change in the glyphosate-binding site, shifting Thr(97) and Gly(96) toward the inhibitor molecule. We conclude that the decreased inhibitory potency observed for glyphosate is a result of these mutation-induced long-range structural changes. The implications of our findings concerning the development and spread of glyphosate-resistant weeds are discussed.