Mechanistic Diversity and Functional Roles Define the Substrate Specificity and Ligand Binding of Bacterial PGP Phosphatases.

Abstract

Phosphatidylglycerol (PG) 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 PG at wild-type 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.