Preferential Hydrolysis of Aberrant Intermediates by the Type II Thioesterase in Escherichia coli Nonribosomal Enterobactin Synthesis: Substrate Specificities and Mutagenic Studies on the Active-Site Residues
Preferential Hydrolysis of Aberrant Intermediates by the Type II Thioesterase in Escherichia coli Nonribosomal Enterobactin Synthesis: Substrate Specificities and Mutagenic Studies on the Active-Site Residues
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DOI：
10.1021/bi802165x
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发表时间：
2009-03-03
期刊：
影响因子：
2.9
通讯作者：
Guo, Zhihong
中科院分区：
文献类型：
--
作者：
Guo, Zu-Feng;Sun, Yueru;Guo, Zhihong
II型硫酯酶EntH是大肠杆菌中肠杆菌素的最佳非核糖体生物合成所需的热狗折叠蛋白。其在生物合成中的校正活性通过其在体外被同源前体2，3-二羟基苯甲酸酯的类似物阻断的肠杆菌素合成的有效恢复来证实。稳态动力学研究表明，EntH识别磷酸泛酰巯基乙胺基团和其硫酯底物的芳基部分中的羟基化模式。值得注意的是，它能够区分异常的中间体从正常的肠杆菌素装配线，通过证明至少10倍高的催化效率对硫酯衍生自异常芳基前体没有对羟基，如水杨酸酯。通过结构比较和定点诱变，发现硫酯酶具有与来自节杆菌属菌株SU的4-羟基苯甲酰-CoA硫酯酶非常相似的活性位点，并且与所有其它热狗硫酯酶一样涉及酸性残基（谷氨酸-63）作为催化碱或亲核试剂。此外，EntH对底物羟基化模式的特异性被发现依赖于活性位点组氨酸-54，苏氨酸-64，丝氨酸-67和甲硫氨酸-68，当它们被丙氨酸单独取代时，选择性显着降低甚至逆转。这些残基可能负责酶与底物的差异相互作用，这导致肠杆菌素组装线中正常和异常前体之间的区别。这些结果表明，II型硫酯酶发展其独特的能力，以识别异常的中间体从通用的催化平台的热狗蛋白，并建议在非核糖体肽合成的11型硫酯酶的主动搜索机制。
The type II thioesterase EntH is a hotdog fold protein required for optimal nonribosomal biosynthesis of enterobactin in Escherichia coli. Its proposed proofreading activity in the biosynthesis is confirmed by its efficient restoration of enterobactin synthesis blocked in vitro by analogs of the cognate precursor 2,3-dihydroxybenzoate. Steady-state kinetic studies show that EntH recognizes the phosphopantetheine group and the pattern of hydroxylation in the aryl moiety of its thioester substrates. Remarkably, it is able to distinguish aberrant intermediates from the normal one in the enterobactin assembly line by demonstrating at least 10-fold higher catalytic efficiency toward thioesters derived from aberrant aryl precursors without a para-hydroxyl group, such as salicylate. By structural comparison and site-directed mutagenesis, the thioesterase is found to possess an active site closely resembling that of the 4-hydroxybenzoyi-CoA thioesterase from Arthrobacter sp. strain SU and to involve an acidic residue (glutamate-63) as the catalytic base or nucleophile like all other hotdog thioesterases. In addition, the EntH specificities toward the substrate hydroxylation pattern are found to depend on the active-site histidine-54, threonine-64, serine-67, and methionine-68 with the selectivity significantly reduced or even reversed when they are individually replaced by alanine. These residues are likely responsible for differential interaction of the enzyme with the substrates which leads to distinction between the normal and aberrant precursors in the enterobactin assembly line. These results show that the type II thioesterase evolves its distinctive ability to recognize the aberrant intermediates from the versatile catalytic platform of hotdog proteins and suggests an active search mechanism for type 11 thioesterases in nonribosomal peptide synthesis.