Among the proteins required for lipid metabolism in Mycobacterium tuberculosis are a significant number of uncharacterized serine hydrolases, especially lipases and esterases. Using a streamlined synthetic method, a library of immolative fluorogenic ester substrates was expanded to better represent the natural lipidomic diversity of Mycobacterium. This expanded fluorogenic library was then used to rapidly characterize the global structure activity relationship (SAR) of mycobacterial serine hydrolases in M. smegmatis under different growth conditions. Confirmation of fluorogenic substrate activation by mycobacterial serine hydrolases was performed using nonspecific serine hydrolase inhibitors and reinforced the biological significance of the SAR. The hydrolases responsible for the global SAR were then assigned using gel-resolved activity measurements, and these assignments were used to rapidly identify the relative substrate specificity of previously uncharacterized mycobacterial hydrolases. These measurements provide a global SAR of mycobacterial hydrolase activity, a picture of cycling hydrolase activity, and a detailed substrate specificity profile for previously uncharacterized hydrolases.

中文翻译：

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使用荧光酯底物库测量分枝杆菌丝氨酸水解酶的整体底物特异性

在结核分枝杆菌中脂质代谢所需的蛋白质中，有大量未表征的丝氨酸水解酶，特别是脂肪酶和酯酶。使用简化的合成方法，扩大了具有消炎作用的含氟酯底物的文库，以更好地代表分枝杆菌的天然脂质组学多样性。然后使用这个扩展的荧光文库快速表征耻垢分枝杆菌中分枝杆菌丝氨酸水解酶的整体结构活性关系（SAR）在不同的生长条件下。使用非特异性丝氨酸水解酶抑制剂进行了分枝杆菌丝氨酸水解酶对荧光底物激活的确认，并增强了SAR的生物学意义。然后，使用凝胶分辨活性测量法对负责总体SAR的水解酶进行分配，并将这些分配用于快速鉴定以前未表征的分枝杆菌水解酶的相对底物特异性。这些测量提供了分枝杆菌水解酶活性的整体SAR，循环水解酶活性的图片以及以前未表征的水解酶的详细底物特异性谱。