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首页> 外文期刊>Journal of Molecular Biology >Crystal structure of Escherichia coli enterobactin-specific isochorismate synthase (EntC) bound to its reaction product isochorismate: implications for the enzyme mechanism and differential activity of chorismate-utilizing enzymes.
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Crystal structure of Escherichia coli enterobactin-specific isochorismate synthase (EntC) bound to its reaction product isochorismate: implications for the enzyme mechanism and differential activity of chorismate-utilizing enzymes.

机译：大肠埃希氏杆菌特异性肠异actorismate合成酶（EntC）的晶体结构绑定到其反应产物isochoorismate：酶机制和分支酸利用酶的差异活性的含义。

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摘要

EntC, one of two isochorismate synthases in Escherichia coli, is specific to the biosynthesis of the siderophore enterobactin. Here, we report the crystal structure of EntC in complex with isochorismate and Mg(2+)at 2.3 A resolution, the first structure of a chorismate-utilizing enzyme with a non-aromatic reaction product. EntC exhibits a complex alpha+beta fold like the other chorismate-utilizing enzymes, such as salicylate synthase and anthranilate synthase. Comparison of active site structures allowed the identification of several residues, not discussed previously, that might be important for the isochorismate activity of the EntC. Although EntC, MenF and Irp9 all convert chorismate to isochorismate, only Irp9 subsequently exhibits isochorismate pyruvate lyase activity resulting in the formation of salicylate and pyruvate as the reaction products. With a view to understanding the roles of these amino acid residues in the conversion of chorismate to isochorismate and to obtaining clues about the pyruvate lyase activity of Irp9, several mutants of EntC were generated in which the selected residues in EntC were substituted for those of Irp9: these included A303T, L304A, F327Y, I346L and F359Q mutations. Biochemical analysis of these mutants indicated that the side chain of A303 in EntC may be crucial in the orientation of the carbonyl to allow formation of a hydrogen bond with isochorismate. Some mutations, such as L304A and F359Q, give rise to a loss of catalytic activity, whereas others, such as F327Y and I346L, show that subtle changes in the otherwise closely similar active sites influence activity. We did not find a combination of these residues that conferred pyruvate lyase activity.
机译：EntC是大肠埃希氏菌中两种等渗酸合成酶之一，对铁载体肠杆菌素的生物合成具有特异性。在这里，我们报告了EntC的晶体结构，其与异方酸和Mg（2+）的复合物为2.3 A分辨率，这是一种利用分支酸酯的酶与非芳香族反应产物的第一种结构。 EntC与其他利用分支酸盐的酶（例如水杨酸合酶和邻氨基苯甲酸合酶）一样，表现出复杂的α+β折叠。活性位点结构的比较允许鉴定几个之前未讨论过的残基，这可能对EntC的等渗活性很重要。尽管EntC，MenF和Irp9都将分支酸盐转化为异硬脂酸，但只有Irp9随后显示出异硬脂酸丙酮酸裂解酶活性，导致水杨酸酯和丙酮酸作为反应产物形成。为了了解这些氨基酸残基在分支酸向异麦酸酯转化中的作用并获得有关Irp9丙酮酸裂解酶活性的线索，生成了EntC的几个突变体，其中EntC中的选定残基替代了Irp9的残基。 ：包括A303T，L304A，F327Y，I346L和F359Q突变。这些突变体的生化分析表明，EntC中A303的侧链可能对羰基的取向至关重要，以允许与异邻苯二甲酸酯形成氢键。一些突变（例如L304A和F359Q）会导致催化活性的丧失，而其他一些突变（例如F327Y和I346L）则表明，在其他方面极为相似的活性位点中的细微变化会影响活性。我们没有发现赋予丙酮酸裂解酶活性的这些残基的组合。

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