PlsX is the first enzyme in the pathway that produces phosphatidic acid in Gram-positive bacteria. It makes acylphosphate from acyl-acyl carrier protein (acyl-ACP) and is also involved in coordinating phospholipid and fatty acid biosyntheses. PlsX is a peripheral membrane enzyme in Bacillus subtilis, but how it associates with the membrane remains largely unknown. In the present study, using fluorescence microscopy, liposome sedimentation, differential scanning calorimetry, and acyltransferase assays, we determined that PlsX binds directly to lipid bilayers and identified its membrane anchoring moiety, consisting of a hydrophobic loop located at the tip of two amphipathic dimerization helices. To establish the role of the membrane association of PlsX in acylphosphate synthesis and in the flux through the phosphatidic acid pathway, we then created mutations and gene fusions that prevent PlsX's interaction with the membrane. Interestingly, phospholipid synthesis was severely hampered in cells in which PlsX was detached from the membrane, and results from metabolic labeling indicated that these cells accumulated free fatty acids. Because the same mutations did not affect PlsX transacylase activity, we conclude that membrane association is required for the proper delivery of PlsX's product to PlsY, the next enzyme in the phosphatidic acid pathway. We conclude that PlsX plays a dual role in phospholipid synthesis, acting both as a catalyst and as a chaperone protein that mediates substrate channeling into the pathway.

中文翻译：

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磷脂酸途径酶PlsX在细菌磷脂合成中同时起催化和引导作用。

PlsX是该途径中第一个在革兰氏阳性细菌中产生磷脂酸的酶。它由酰基-酰基载体蛋白（酰基-ACP）制得酰基磷酸酯，还参与协调磷脂和脂肪酸的生物合成。PlsX是枯草芽孢杆菌中的外周膜酶，但是如何与膜结合仍然非常未知。在本研究中，我们使用荧光显微镜，脂质体沉淀，差示扫描量热法和酰基转移酶测定方法，确定PlsX直接与脂质双层结合，并鉴定了其膜锚定部分，该膜锚定部分由位于两个两亲性二聚螺旋末端的疏水环组成。 。要确定PlsX的膜缔合在酰基磷酸合成中以及在通过磷脂酸途径的通量中的作用，然后，我们创建了阻止PlsX与膜相互作用的突变和基因融合。有趣的是，磷脂酶在PlsX从膜上脱落的细胞中严重受阻，代谢标记的结果表明这些细胞积累了游离脂肪酸。因为相同的突变不会影响PlsX转酰酶的活性，所以我们得出结论，膜缔合是将PlsX的产物正确递送到磷脂酸途径中的下一个酶PlsY所必需的。我们得出的结论是，PlsX在磷脂合成中起双重作用，既充当催化剂，又充当介导底物通道进入通道的伴侣蛋白。PlsX从膜上脱落的细胞中，磷脂的合成受到严重阻碍，代谢标记的结果表明这些细胞积累了游离脂肪酸。因为相同的突变不会影响PlsX转酰酶的活性，所以我们得出结论，膜缔合是将PlsX的产物正确递送到磷脂酸途径中的下一个酶PlsY所必需的。我们得出的结论是，PlsX在磷脂合成中起双重作用，既充当催化剂，又充当介导底物通道进入通道的伴侣蛋白。PlsX从膜上脱落的细胞中，磷脂的合成受到严重阻碍，代谢标记的结果表明这些细胞积累了游离脂肪酸。因为相同的突变不会影响PlsX转酰酶的活性，所以我们得出结论，膜缔合是将PlsX的产物正确递送到磷脂酸途径中的下一个酶PlsY所必需的。我们得出的结论是，PlsX在磷脂合成中起双重作用，既充当催化剂，又充当介导底物通道进入通道的伴侣蛋白。我们得出结论，膜结合是将PlsX的产物正确递送到PlsY（磷脂酸途径中的下一个酶）所必需的。我们得出的结论是，PlsX在磷脂合成中起双重作用，既充当催化剂，又充当介导底物通道进入通道的伴侣蛋白。我们得出结论，膜结合是将PlsX的产物正确递送到PlsY（磷脂酸途径中的下一个酶）所必需的。我们得出的结论是，PlsX在磷脂合成中起双重作用，既充当催化剂，又充当介导底物通道进入通道的伴侣蛋白。