Crystal structure of lipid phosphatase Escherichia coli phosphatidylglycerophosphate phosphatase B

Abstract

Membrane-integrated type II phosphatidic acid phosphatases (PAP2s) are important for numerous bacterial to human biological processes, including glucose transport, lipid metabolism, and signaling. Escherichia coli phosphatidylglycerol-phosphate phosphatase B (ecPgpB) catalyzes removing the terminal phosphate group from a lipid carrier, undecaprenyl pyrophosphate, and is essential for transport of many hydrophilic small molecules across the membrane. We determined the crystal structure of ecPgpB at a resolution of 3.2 Å. This structure shares a similar folding topology and a nearly identical active site with soluble PAP2 enzymes. However, the substrate binding mechanism appears to be fundamentally different from that in soluble PAP2 enzymes. In ecPgpB, the potential substrate entrance to the active site is located in a cleft formed by a V-shaped transmembrane helix pair, allowing lateral movement of the lipid substrate entering the active site from the membrane lipid bilayer. Activity assays of point mutations confirmed the importance of the catalytic residues and potential residues involved in phosphate binding. The structure also suggests an induced-fit mechanism for the substrate binding. The 3D structure of ecPgpB serves as a prototype to study eukaryotic PAP2 enzymes, including human glucose-6-phosphatase, a key enzyme in the homeostatic regulation of blood glucose concentrations.T ype II phosphatidic acid phosphatases (PAP2s) are a large family of phosphatases important for lipid metabolism and signaling (1, 2). PAP2 proteins have been found in all life kingdoms from bacteria to mammals. They catalyze dephosphorylation of broad substrates by specifically hydrolyzing phosphoric monoester bonds. Their substrates include variety of phosphorylated carbohydrates, peptides, and lipids. PAP2s are involved in vesicular trafficking, secretion, and endocytosis (e.g., the enzyme phosphatidate phosphatase APP1 in yeast) (3); protein glycosylation [e.g., dolichyl pyrophosphate phosphatase 1 (DOLPP1) in the mouse] (4); energy storage (e.g., triacylglycerol biosynthesis) (5); and stress response (6). In contrast to type I PAP enzymes, which are Mg 2+ -dependent and usually soluble, PAP2 proteins are Mg 2+ -independent, and many of PAP2 enzymes are integral transmembrane (TM) proteins (7). Whereas the soluble branch of PAP2s is called class A nonspecific acid phosphatases (NSAPs) (8), the TM branch of the PAP2 family is also called the lipid phosphatase/phosphotransferase family (2) or lipid phosphate phosphatase family (9). Human glucose-6-phosphatase (G6Pase), the key enzyme in the homeostatic regulation of blood glucose concentrations, belongs to the TM PAP2 subfamily (10). Thus, the TM property is unique to the PAP2 family.In Escherichia coli, undecaprenyl phosphate (C 55 -P), a 55-carbon single-lipid chain phospholipid, serves as a carrier lipid to transfer a variety of phosphate-linked polymers across the periplasmic membrane from the cytosol to the periplasmic space. Recently, the crystal structure of phospho-N-acet...

Lipid phosphate phosphatases and their roles in mammalian physiology and pathology

“…However, the orientation of the catalytic site and mechanism of action have been postulated through a combination of computational modeling and the crystallographic structure of a related enzyme, chloroperoxidase (31)(32)(33). Chloroperoxidase is a soluble enzyme, and a better understanding of the topology of the LPPs can be gained from the crystal structure of phosphatidylglycerolphosphate phosphatase B from Eshcherichia coli , which, like the LPPs, is an integral membrane protein ( 34 ). The LPPs possess six transmembrane ␣ -helices and when located in the plasma membrane, the C-and N-termini face the cytoplasmic side and the three catalytic domains face the extracellular side ( Fig.…”

Impact of Membrane Phospholipid Alterations in Escherichia coli on Cellular Function and Bacterial Stress Adaptation

“…Phospholipid metabolism has not been explored using pharmacological modulation, possibly due to the lack of comprehensive structure-function studies on phospholipid biosynthetic enzymes and the availability of only a few high-resolution structures (16)(17)(18)(19). Additionally, current screens performed on single-knockout strain libraries (20,21) often do not include genes involved in phospholipid biosynthesis pathways (such as cds, pss, psd, or pgs) (Fig.…”

Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

“…BacA provides 75% of the cell's UPP-Pase activity, and overexpression of BacA makes cells bacitracin resistant (7). PgpB was originally identified in mutant cells lacking phosphatidylglycerol phosphate phosphatase activity (24) and has been shown to have broad substrate specificity (25,26). The BacA, YbjG, and PgpB enzymes are functionally redundant; single mutants lacking any one of the three genes do not show significant growth defects.…”