Kinetic and Inhibition Studies of Dihydroxybenzoate-AMP Ligase from Escherichia coli
Kinetic and Inhibition Studies of Dihydroxybenzoate-AMP Ligase from Escherichia coli
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DOI：
10.1021/bi100350c
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发表时间：
2010-05-04
期刊：
影响因子：
2.9
通讯作者：
Blanchard, John S.
中科院分区：
文献类型：
--
作者：
Sikora, Alison L.;Wilson, Daniel J.;Blanchard, John S.
抑制病原菌铁载体生物合成途径是抗菌药物开发的一个有前途的策略。大肠杆菌在细胞内缺铁时，可合成分泌铁载体肠杆菌素。在这里，我们描述了一个详细的动力学分析EntE，肠杆菌素合成酶基因簇中的六种酶之一。EntE催化2，3-二羟基苯甲酸（DHB）和磷酸泛酰巯基乙胺化的EntB（holo-EntB）的ATP依赖性缩合以形成共价芳基化的EntB，该产物对于肠杆菌素的最终组装至关重要。初速度的研究表明，EntE收益通过双-单-单-双乒乓动力学机制与k（猫）等于2.8 s（-1）和Km值为2.5，430，和2.9 μ M的DHB，ATP，和holo-EntB-ArCP，分别。抑制和直接结合实验表明，在第一个半反应（腺苷酸化），DHB首先结合到游离酶，然后是ATP和焦磷酸盐的释放，形成腺苷酸中间体。在第二个半反应（连接）期间，磷酸泛酰巯基乙胺化的EntB与酶结合，随后释放产物AMP和芳基化的EntB。两种水解稳定的腺苷酸类似物，5 '-O-[N-（水杨基）氨磺酰基]腺苷（Sal-AMS）和5'-O-[N-（2，3-二羟基苯甲酰基）氨磺酰基]腺苷（DHB-AMS），被证明是该酶的缓慢起效的紧密结合抑制剂，K-app（i）值分别为0.9和3.8 nM。直接结合实验，通过等温滴定量热法，揭示低皮摩尔的解离常数，这两个类似物相对于EntE。Sal-AMS和DHB-AMS与EntE的紧密结合表明这些化合物可以进一步开发为靶向该酶的有效抗生素。
Inhibition of siderophore biosynthetic pathways in pathogenic bacteria represents a promising strategy for antibacterial drug development. Escherichia call synthesize and secrete the small molecule iron chelator siderophore, enterobactin, in response to intracellular iron depletion. Here we describe a detailed kinetic analysis of EntE, one of six enzymes in the enterobactin synthetase gene cluster. EntE catalyzes the ATP-dependent condensation of 2,3-dihydroxybenzoic acid (DHB) and phosphopantetheinylated EntB (holo-EntB) to form covalently arylated EntB, a product that is vital for the final assembly of enterobactin. Initial velocity studies show that EntE proceeds via a bi-uni-uni-bi ping-pong kinetic mechanism with a k(cat) equal to 2.8 s(-1) and Km values of 2.5, 430, and 2.9 mu M for DHB, ATP, and holo-EntB-ArCP, respectively. Inhibition and direct binding experiments suggest that, during the first half-reaction (adenylation), DHB binds first to the free enzyme, followed by ATP and the release of pyrophosphate to form the adenylate intermediate. During the second half-reaction (ligation), phosphopantetheinylated EntB binds to the enzyme followed by the release of products, AMP and arylated EntB. Two hydrolytically stable adenylate analogues, 5'-O[N-(salicyl)sulfamoyl]adenosine (Sal-AMS) and 5'-O-[N-(2,3-dihydroxybenzoyl)sulfamoyl]adenosine (DHB-AMS), are shown to act as slow-onset tight-binding inhibitors of the enzyme with K-app(i) values of 0.9 and 3.8 nM, respectively. Direct binding experiments, via isothermal titration calorimetry, reveal low picomolar dissociation constants for both analogues with respect to EntE. The tight binding of Sal-AMS and DHB-AMS to EntE suggests that these compounds may be developed further as effective antibiotics targeted to this enzyme.