Trehalose is an essential disaccharide for mycobacteria and a key constituent of several cell wall glycolipids with fundamental roles in pathogenesis. Mycobacteria possess two pathways for trehalose biosynthesis. However, only the OtsAB pathway was found to be essential in Mycobacterium tuberculosis, with marked growth and virulence defects of OtsA mutants and strict essentiality of OtsB2. Here, we report the first mycobacterial OtsA structures from Mycobacterium thermoresistibile in both apo and ligand-bound forms. Structural information reveals three key residues in the mechanism of substrate preference that were further confirmed by site-directed mutagenesis. Additionally, we identify 2-oxoglutarate and 2-phosphoglycerate as allosteric regulators of OtsA. The structural analysis in this work strongly contributed to define the mechanisms for feedback inhibition, show different conformational states of the enzyme, and map a new allosteric site.IMPORTANCE Mycobacterial infections are a significant source of mortality worldwide, causing millions of deaths annually. Trehalose is a multipurpose disaccharide that plays a fundamental structural role in these organisms as a component of mycolic acids, a molecular hallmark of the cell envelope of mycobacteria. Here, we describe the first mycobacterial OtsA structures. We show mechanisms of substrate preference and show that OtsA is regulated allosterically by 2-oxoglutarate and 2-phosphoglycerate at an interfacial site. These results identify a new allosteric site and provide insight on the regulation of trehalose synthesis through the OtsAB pathway in mycobacteria.

中文翻译：

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分枝杆菌的OtsA结构揭示了底物偏好机制和2-氧戊二酸酯和2-磷酸甘油酸酯的变构调节。

海藻糖是分枝杆菌必不可少的二糖，是几种细胞壁糖脂的关键成分，在发病机理中起着重要作用。分枝杆菌具有海藻糖生物合成的两个途径。但是，只有OtsAB途径在结核分枝杆菌中被发现是必需的，具有明显的OtsA突变体生长和毒力缺陷以及OtsB2的严格必需性。在这里，我们报告了载脂蛋白和配体结合形式的热分枝杆菌的第一个分枝杆菌OtsA结构。结构信息揭示了底物偏好机制中的三个关键残基，这些残基已通过定点诱变得到进一步证实。此外，我们确定2-氧戊二酸和2-磷酸甘油酸为OtsA的变构调节剂。这项工作的结构分析为定义反馈抑制机制，显示酶的不同构象状态以及定位新的变构位点做出了重要贡献。重要事项分枝杆菌感染是全世界重要的死亡来源，每年造成数百万人死亡。海藻糖是一种多用途的二糖，在这些生物中作为分枝杆菌酸（分枝杆菌细胞包膜的分子标志）的一种成分发挥着基本的结构作用。在这里，我们描述了第一个分枝杆菌的OtsA结构。我们显示了底物偏好的机制，并表明OtsA是由2-氧戊二酸酯和2-磷酸甘油酸酯在界面部位变构调节的。