Phosphatidylglycerol is a critical membrane phospholipid in microorganisms, synthesized via the dephosphorylation of phosphatidylglycerol-phosphate (PGP) by three membrane-bound phosphatases: PgpA, PgpB, and PgpC. While any one of these enzymes can produce phosphatidylglycerol at WT levels, the reason for the presence of all three in bacteria remains unclear. To address this question, we characterized these phosphatases in vitro to uncover their mechanistic differences. Our assays demonstrated that all three enzymes catalyze the hydrolysis of PGP but exhibit distinct substrate selectivity. PgpB displays a broad substrate range, dephosphorylating various lipid phosphates, while PgpA and PgpC show a higher specificity for lysophosphatidic acid and PGP. Notably, PgpA also effectively dephosphorylates soluble metabolites, such as glycerol-3-phosphate and glyceraldehyde-3-phosphate, suggesting its unique substrate-binding mechanism that relies on precise recognition of the glycerol head group rather than the fatty acid. Inhibitor screening with synthetic substrate analogs revealed that PgpB is inhibited by lipid-like compounds XY-14 and XY-55, whereas PgpA and PgpC are unaffected. Structural analysis and mutational studies identified two charged residues at the catalytic site entry for inhibitor binding in PgpB and support the notion that the PgpB maintains a large substrate binding site to accommodate multiple ligand binding conformations. These findings underscore the distinct substrate recognition mechanisms and possible functional roles of PgpA, PgpB, and PgpC in bacterial lipid metabolism and offer insights for developing novel inhibitors targeting bacterial membrane phospholipid biosynthesis.

中文翻译：

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机制多样性和功能作用决定了细菌 PGP 磷酸酶的底物特异性和配体结合


磷脂酰甘油是微生物中一种重要的膜磷脂，通过三种膜结合磷酸酶（PgpA、PgpB 和 PgpC）对磷脂酰甘油磷酸 （PGP） 的去磷酸化而合成。虽然这些酶中的任何一种都可以产生 WT 水平的磷脂酰甘油，但细菌中存在这三种酶的原因尚不清楚。为了解决这个问题，我们在体外表征了这些磷酸酶 ，以揭示它们的机制差异。我们的分析表明，所有三种酶都催化 PGP 的水解，但表现出不同的底物选择性。PgpB 显示出广泛的底物范围，使各种脂质磷酸盐去磷酸化，而 PgpA 和 PgpC 对溶血磷脂酸和 PGP 表现出更高的特异性。值得注意的是，PgpA 还有效地使可溶性代谢物（如 3-磷酸甘油和甘油醛-3-磷酸）去磷酸化，这表明其独特的底物结合机制依赖于对甘油头部基团而不是脂肪酸的精确识别。用合成底物类似物筛选抑制剂显示，PgpB 受脂质样化合物 XY-14 和 XY-55 抑制，而 PgpA 和 PgpC 不受影响。结构分析和突变研究在 PgpB 中抑制物结合的催化位点入口处确定了两个带电残基，并支持 PgpB 保持大底物结合位点以适应多种配体结合构象的观点。这些发现强调了 PgpA、PgpB 和 PgpC 在细菌脂质代谢中独特的底物识别机制和可能的功能作用，并为开发靶向细菌膜磷脂生物合成的新型抑制剂提供了见解。