In mycobacteria, phosphatidylinositol (PI) acts as a common lipid anchor for key components of the cell wall, including the glycolipids phosphatidylinositol mannoside, lipomannan, and lipoarabinomannan. Glycolipids in Mycobacterium tuberculosis, the causative agent of tuberculosis, are important virulence factors that modulate the host immune response. The identity-defining step in PI biosynthesis in prokaryotes, unique to mycobacteria and few other bacterial species, is the reaction between cytidine diphosphate–diacylglycerol and inositol-phosphate to yield phosphatidylinositol-phosphate, the immediate precursor to PI. This reaction is catalyzed by the cytidine diphosphate–alcohol phosphotransferase phosphatidylinositol-phosphate synthase (PIPS), an essential enzyme for mycobacterial viability. Here we present structures of PIPS from Mycobacterium kansasii with and without evidence of donor and acceptor substrate binding obtained using a crystal engineering approach. PIPS from Mycobacterium kansasii is 86% identical to the ortholog from M. tuberculosis and catalytically active. Functional experiments guided by our structural results allowed us to further characterize the molecular determinants of substrate specificity and catalysis in a new mycobacterial species. This work provides a framework to strengthen our understanding of phosphatidylinositol-phosphate biosynthesis in the context of mycobacterial pathogens.

在分枝杆菌中，磷脂酰肌醇 (PI) 充当细胞壁关键成分的常见脂质锚，包括糖脂磷脂酰肌醇甘露糖苷、脂甘露聚糖和脂阿拉伯甘露聚糖。结核分枝杆菌中的糖脂结核病的病原体是调节宿主免疫反应的重要毒力因子。原核生物中 PI 生物合成的身份定义步骤是分枝杆菌和少数其他细菌物种独有的，是胞苷二磷酸二酰甘油和肌醇磷酸之间的反应，产生磷脂酰肌醇磷酸，这是 PI 的直接前体。该反应由胞苷二磷酸醇磷酸转移酶磷脂酰肌醇磷酸合酶 (PIPS) 催化，PIPS 是分枝杆菌生存力的必需酶。在这里，我们展示了来自堪萨斯分枝杆菌的 PIPS 结构，有或没有使用晶体工程方法获得的供体和受体底物结合的证据。来自堪萨斯分枝杆菌的PIPS与来自结核分枝杆菌的直系同源物有 86% 相同，并且具有催化活性。以我们的结构结果为指导的功能实验使我们能够进一步表征新分枝杆菌物种中底物特异性和催化作用的分子决定因素。这项工作提供了一个框架，以加强我们对分枝杆菌病原体背景下磷脂酰肌醇-磷酸酯生物合成的理解。