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.
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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DOI：
10.1016/j.jmb.2010.01.019
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发表时间：
2010-03
影响因子：
5.6
通讯作者：
S. Sridharan;N. Howard;O. Kerbarh;M. Błaszczyk;C. Abell;T. Blundell
中科院分区：
文献类型：
--
作者：
S. Sridharan;N. Howard;O. Kerbarh;M. Błaszczyk;C. Abell;T. Blundell
EntC是大肠杆菌中两种异分支酸脱氢酶之一，对铁载体肠杆菌素的生物合成具有特异性。在这里，我们报告的晶体结构的EntC在复杂的异分支酸和Mg 2+在2.3倍分辨率，第一个结构的分支酸利用酶与非芳香族反应产物。EntC表现出复杂的α+β折叠，像其他分支酸利用酶，如水杨酸合酶和邻氨基苯甲酸合酶。活性位点结构的比较允许几个残基的识别，没有讨论过，这可能是重要的异分支活性的EntC。虽然EntC、MenF和Irp9都将分支酸转化为异分支酸，但只有Irp9随后表现出异分支酸丙酮酸裂解酶活性，导致形成水杨酸和丙酮酸作为反应产物。为了理解这些氨基酸残基在分支酸转化为异分支酸中的作用，并获得关于Irp9的丙酮酸裂解酶活性的线索，产生了几种EntC突变体，其中EntC中的选定残基取代了Irp9的那些：这些突变体包括A303T、L304A、F327Y、I346L和F359Q突变。对这些突变体的生化分析表明，EntC中A303的侧链在羰基的取向中可能是至关重要的，以允许与异分支酸形成氢键。一些突变，如L304A和F359Q，导致催化活性的丧失，而其他突变，如F327Y和I346L，表明在其他方面非常相似的活性位点的细微变化影响活性。我们没有发现赋予丙酮酸裂解酶活性的这些残基的组合。
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 Mg2+at 2.3 Å resolution, the first structure of a chorismate-utilizing enzyme with a non-aromatic reaction product. EntC exhibits a complex α+β 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.