Biosynthesis of a water-soluble lipid I analogue and a convenient assay for translocase I.
Biosynthesis of a water-soluble lipid I analogue and a convenient assay for translocase I.
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水溶性脂质 I 类似物的生物合成和易位酶 I 的便捷测定。
DOI：
10.1016/j.ab.2014.05.018
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发表时间：
2014
影响因子：
2.9
通讯作者：
Kurosu,Michio
中科院分区：
文献类型：
--
作者：
Siricilla,Shajila;Mitachi,Katsuhiko;Skorupinska-Tudek,Karolina;Swiezewska,Ewa;Kurosu,Michio
转位酶I（MraY/MurX）是绝大多数细菌生长中的必需酶，其催化从UDP-MurNAc-五肽（Park核苷酸）转化为异戊二烯基-MurNAc-五肽（脂质I），异戊二烯基-MurNAc-五肽（脂质I）是第一个膜锚定肽聚糖前体。MurX在新型结核病（TB）药物的开发中受到了相当大的关注，因为MurX抑制剂比临床使用的TB药物更快地杀死呈指数增长的结核分枝杆菌（Mtb）。从Mtb中分离出的脂质I含有C50-异戊烯基单元，其水溶性非常差;因此，脂质I的这种化学特征使得MurX酶测定法无法用于筛选，并且缺乏酶测定的重现性。我们已经建立了一个可扩展的化学合成Park的核苷酸-N ε-丹磺酰硫脲2，可用作MurX酶底物，形成脂质I类似物。在我们的研究中的异戊二烯基磷酸在MraY/MurX催化的脂质I类似物的合成与2的最低结构要求，我们发现，橙花基磷酸（C10磷酸）可以识别的MraY/MurX生成的水溶性脂质I类似物在优化的条件下，以定量的产量。在这里，我们报告了一种快速和强大的分析方法，用于定量MraY/MurX抑制活性的库分子。
Translocase I (MraY/MurX) is an essential enzyme in growth of the vast majority of bacteria that catalyzes the transformation from UDP-MurNAc-pentapeptide (Park’s nucleotide) to prenyl-MurNAc-pentapeptide (lipid I), the first membrane-anchored peptidoglycan precursor. MurX has received considerable attention in the development of new tuberculosis (TB) drugs due to the fact that the MurX inhibitors kill exponentially growingMycobacterium tuberculosis(Mtb) much faster than clinically used TB drugs. Lipid I isolated fromMtbcontains the C50-prenyl unit that shows very poor water solubility; thus, this chemical characteristic of lipid I renders MurX enzyme assays impractical for screening and lacks reproducibility of the enzyme assays. We have established a scalable chemical synthesis of Park’s nucleotide-Nε-dansylthiourea2that can be used as a MurX enzymatic substrate to form lipid I analogues. In our investigation of the minimum structure requirement of the prenyl phosphate in the MraY/MurX-catalyzed lipid I analogue synthesis with2, we found that neryl phosphate (C10phosphate) can be recognized by MraY/MurX to generate the water-soluble lipid I analogue in quantitative yield under the optimized conditions. Here, we report a rapid and robust analytical method for quantifying MraY/MurX inhibitory activity of library molecules.