How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli
How the mutation glycine96 to alanine confers glyphosate insensitivity to 5-enolpyruvyl shikimate-3-phosphate synthase from Escherichia coli
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DOI：
10.1007/s00425-002-0908-0
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发表时间：
2002-11-01
期刊：
影响因子：
4.3
通讯作者：
Schönbrunn, E
中科院分区：
文献类型：
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作者：
Eschenburg, S;Healy, ML;Schönbrunn, E
5-烯醇式莽草酸-3-磷酸（EPSP）合酶（EC 2.5.1.19）对于植物和微生物中芳香族化合物的生物合成是必需的，并且是除草剂草甘膦的独特靶标。最早报道的草甘膦不敏感酶之一是来自肺炎克雷伯氏菌的EPSP合酶的Gly 96 Ala突变体。我们已经将这种单位点突变引入到来自埃希氏菌的高度同源的EPSP合酶中。杆菌突变酶对草甘膦不敏感，对其第一底物莽草酸-3-磷酸（S3 P）的亲和力不变，但对其第二底物磷酸烯醇丙酮酸（PEP）的亲和力降低了30倍。使用X射线晶体学，我们解决了Gly 96 Ala-EPSP合酶与S3 P的结构，分辨率为0.17 nm。晶体结构表明，额外的甲基从Ala 96突出到酶的活性位点。虽然酶和S3 P之间的相互作用不受影响，但草甘膦结合的可及体积大大减少。利用分子建模的晶体学结果，我们证明，PEP，但不是草甘膦可以停靠在Gly 96 Ala修饰的结合位点。预测的PEP结合位点满足早期提出的PEP与EPSP合酶的相互作用模式，并证实了草甘膦和PEP靶向相同结合位点的假设。
The enzyme 5-enolpyruvyl shikimate-3-phosphate (EPSP) synthase (EC 2.5.1.19) is essential for the biosynthesis of aromatic compounds in plants and microbes and is the unique target of the herbicide glyphosate. One of the first glyphosate-insensitive enzymes reported was a Gly96Ala mutant of EPSP synthase from Klebsiella pneumoniae. We have introduced this single-site mutation into the highly homologous EPSP synthase from Escherichia. coli. The mutant enzyme is insensitive to glyphosate with unaltered affinity for its first substrate, shikimate-3-phosphate (S3P), but displays a 30-fold lower affinity for its second substrate, phosphoenolpyruvate (PEP). Using X-ray crystallography, we solved the structure of Gly96Ala-EPSP synthase liganded with S3P to 0.17 nm resolution. The crystal structure shows that the additional methyl group from Ala96 protrudes into the active site of the enzyme. While the interactions between enzyme and S3P remain unaffected, the accessible volume for glyphosate binding is substantially reduced. Exploiting the crystallographic results for molecular modeling, we demonstrate that PEP but not glyphosate can be docked in the Gly96Ala-modified binding site. The predicted PEP binding site satisfies the earlier proposed interaction pattern for PEP with EPSP synthase and corroborates the assumption that glyphosate and PEP target the same binding site.