The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: Development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall
The mycolyltransferase 85A, a putative drug target of Mycobacterium tuberculosis: Development of a novel assay and quantification of glycolipid-status of the mycobacterial cell wall
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DOI：
10.1016/j.mimet.2009.10.010
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发表时间：
2009-12-01
影响因子：
2.2
通讯作者：
Singh, Mahavir
中科院分区：
文献类型：
--
作者：
Elamin, Ayssar A.;Stehr, Matthias;Singh, Mahavir
抗原85复合体(Ag85A、B和C)的酶具有霉菌转移酶活性，并催化合成分枝杆菌细胞壁最丰富的糖脂--脐带因子。脐带因子(海藻糖，6，6‘-二聚乙醇酸，TDM)对于分枝杆菌细胞壁的完整性和芽孢杆菌的致病是必不可少的。因此，TDM生物合成被认为是控制结核分枝杆菌感染的潜在药物靶点。由海藻糖-6‘-单甲酸酯(TMM)的两个分子在抗原85A的作用下合成了海藻糖6，61-二聚甲酸酯(TDM)。我们报告了一种使用天然底物TMM的新的酶分析方法。这种新的比色分析是基于海藻糖降解产生的葡萄糖的定量，海藻糖是抗原85A催化活性的产物。用该方法测定的K-m和K-CAT分别为129.6+/-8.1muM和65.4+/-4.1min(-1)。这一新的方法也适用于针对真菌转移酶(抗原85A)的化合物文库的稳健的高通量筛选(HTS)。该检测方法比目前使用的所有检测方法都要快得多，使用也更方便。该方法在96孔板上的变异系数很低(0.04)，Z‘因子为0.67~0.73，表明该方法具有较高的稳定性。此外，这种新的方法非常适合于分枝杆菌细胞被膜总TMM的定量。(C)2009年，爱思唯尔出版。
The enzymes of the antigen 85 complex (Ag85A, B, and C) possess mycolyltransferase activity and catalyze the synthesis of the most abundant glycolipid of the mycobacterial cell wall, the cord factor. The cord factor (trehalose 6,6'-dimycolate, TDM) is essential for the integrity of the mycobacterial cell wall and pathogenesis of the bacillus. Thus, TDM biosynthesis is regarded as a potential drug target for control of Mycobacterium tuberculosis infections. Trehalose 6,61-dimycolate (TDM) is synthesized from two molecules of trehalose-6'-monomycolate (TMM) by antigen 85A. We report here a novel enzyme assay using the natural substrate TMM. The novel colorimetric assay is based on the quantification of glucose from the degradation of trehalose, which is the product from catalytic activity of antigen 85A. Using the new assay, K-m and K-cat were determined with values of 129.6 +/- 8.1 mu M and 65.4 +/- 4.1 min(-1), respectively. This novel assay is also suitable for robust high-throughput screening (HTS) for compound library screening against mycolyltransferase (antigen 85A). The assay is significantly faster and more convenient to use than all assays currently in use. The assay has a very low coefficient of variance (0.04) in 96-well plates and shows a Z' factor of 0.67-0.73, indicating the robustness of the assay. In addition, this new assay is highly suitable for the quantification of total TMM of the mycobacterial cell envelope. (C) 2009 Published by Elsevier B.V.