Structural Insight into Substrate Selection and Catalysis of Lipid Phosphate Phosphatase PgpB in the Cell Membrane

Abstract

PgpB belongs to the lipid phosphate phosphatase protein family and is one of three bacterial integral membrane phosphatases catalyzing dephosphorylation of phosphatidylglycerol phosphate (PGP) to generate phosphatidylglycerol. Although the structure of its apo form became recently available, the mechanisms of PgpB substrate binding and catalysis are still unclear. We found that PgpB was inhibited by phosphatidylethanolamine (PE) in a competitive mode in vitro Here we report the crystal structure of the lipid-bound form of PgpB. The structure shows that a PE molecule is stabilized in a membrane-embedded tunnel formed by TM3 and the "PSGH" fingerprint peptide near the catalytic site, providing structural insight into PgpB substrate binding mechanism. Noteworthy, in silico docking of varied lipid phosphates exhibited similar substrate binding modes to that of PE, and the residues in the lipid tunnel appear to be important for PgpB catalysis. The catalytic triad in the active site is essential for dephosphorylating substrates lysophosphatidic acid, phosphatidic acid, or sphingosine-1-phosphate but surprisingly not for the native substrate PGP. Remarkably, residue His-207 alone is sufficient to hydrolyze PGP, indicating a specific catalytic mechanism for PgpB in PG biosynthesis. We also identified two novel sensor residues, Lys-93 and Lys-97, on TM3. Our data show that Lys-97 is essential for the recognition of lyso-form substrates. Modification at the Lys-93 position may alter substrate specificity of lipid phosphate phosphatase proteins in prokaryotes versus eukaryotes. These studies reveal new mechanisms of lipid substrate selection and catalysis by PgpB and suggest that the enzyme rests in a PE-stabilized state in the bilayer.

Fat storage-inducing transmembrane (FIT or FITM) proteins are related to lipid phosphatase/phosphotransferase enzymes

“…We found that variation of E-3 reduced the activity of yeast FITMs in all the phenotypes we tested. Where the catalytic triad has been mutagenized more extensively, for example in PgpB, H-2 is essential for all activity, but variation in either H-1 or D-3 affects substrates variably 62 . The natural substrate of PgpB is phosphatidylglycerol phosphate, but it can hydrolyse several other lipid phosphate substrates, which are more sensitive to H 1-A and D 3-A mutations than is the natural ligand.…”

Crystal structure of undecaprenyl-pyrophosphate phosphatase and its role in peptidoglycan biosynthesis

“…It also has pyrophosphatase activity and functions with C55PP as substrate 14 . The structure of E. coli PgpB is known and the enzyme has been shown to have its active site facing the periplasm 14 – 16 . It appears likely therefore that PgpB contributes to the regeneration of C55P from C55PP on the periplasmic side of the membrane.…”

Structural basis for catalysis at the membrane-water interface

“…Mutagenesis designed to occlude the lipid entrance to the active site showed decreased enzyme activity, consistent with the proposed lipid-binding scheme. However, a recently published structure, co-crystallized with phosphatidylethanolamine (PE), shows PE binding in a hydrophobic tunnel formed by TMs 3, 4, and 6, on the other side of the enzyme [38]. PE is not a catalytic substrate of PgpB, but it does seem to act as a competitive inhibitor, suggesting that this binding site may also be utilized by physiological substrates such as UndPP and PA.…”