Substrate Specificity and Membrane Topology of Escherichia coli PgpB, an Undecaprenyl Pyrophosphate Phosphatase

Abstract

The synthesis of the lipid carrier undecaprenyl phosphate (C 55 -P) requires the dephosphorylation of its precursor, undecaprenyl pyrophosphate (C 55 -PP). The latter lipid is synthesized de novo in the cytosol and is also regenerated after its release from the C 55 -PP-linked glycans in the periplasm. In Escherichia coli the dephosphorylation of C 55 -PP was shown to involve four integral membrane proteins, BacA, and three members of the type 2 phosphatidic acid phosphatase family, PgpB, YbjG, and YeiU. Here, the PgpB protein was purified to homogeneity, and its phosphatase activity was examined. This enzyme was shown to catalyze the dephosphorylation of C 55 -PP with a relatively low efficiency compared with diacylglycerol pyrophosphate and farnesyl pyrophosphate (C 15 -PP) lipid substrates. However, the in vitro C 55 -PP phosphatase activity of PgpB was specifically enhanced by different phospholipids. We hypothesize that the phospholipids are important determinants to ensure proper conformation of the atypical long axis C 55 carrier lipid in membranes. Furthermore, a topological analysis demonstrated that PgpB contains six transmembrane segments, a large periplasmic loop, and the type 2 phosphatidic acid phosphatase signature residues at a periplasmic location.Undecaprenyl phosphate (C 55 -P) 2 is a 55-carbon-long polyprenol (see Fig. 1). It is an essential bacterial lipid required for the synthesis of various cell wall polymers such as peptidoglycan, lipopolysaccharides, teichoic acids, osmo-regulated periplasmic glucans, capsular polysaccharides, and the enterobacterial common antigen (1-10). C 55 -P is utilized as a carrier lipid that allows the transport of the hydrophilic oligosaccharide precursors across the cytoplasmic membrane toward the periplasm where the elongation of the glycan chains takes place. Accordingly, the precursor is linked to the carrier lipid via a pyrophosphate linkage (C 55 -PP-substrate) through the action of a specific glycosyltransferase at the cytosolic side of the inner membrane; thereafter, the complex is translocated through the membrane by a yet unknown mechanism, and finally, the glycosyl moiety is transferred to the appropriate expanding polymer. De novo synthesis of C 55 -P implicates two enzymatic steps (11, 12); it originates from undecaprenyl pyrophosphate (C 55 -PP), itself being synthesized by successive condensations of eight isopentenyl pyrophosphates (C 5 -PP) with farnesyl pyrophosphate (C 15 -PP) (Fig. 1) catalyzed by the cytosolic UppS enzyme, a cis-prenyl-pyrophosphate synthase (13, 14). The C 55 -PP must then be dephosphorylated to yield the active monophosphate form of the carrier lipid (11). C 55 -PP is not solely generated by de novo synthesis, but it is also released and recycled after the transfer of the oligosaccharide unit to the growing polymer in the periplasm. It is yet unclear on which side of the membrane C 55 -PP dephosphorylation occurs and how the carrier lipid is translocated across the membrane before being reused.The enzymatic dephosphoryl...

Conservation of the PTEN catalytic motif in the bacterial undecaprenyl pyrophosphate phosphatase, BacA/UppP

“…Interestingly, bacteria with disruption of all three PGP phosphatase genes, pgpA, pgpB and pgpC (Lu et al, 2011), are not viable. However, Touzé et al (2008a) have argued that PgpB functions as a periplasmic undecaprenyl pyrophosphate phosphatase even though their data demonstrates a 100-fold higher activity towards PGP. The basis for their assumption comes from a fourfold enhancement of PgpB activity on undecaprenyl pyrophosphate in the presence of phospholipids.…”

“…Of particular note, based on complete BLAST searches, obvious homologues/orthologues of YbjG and YeiU are not found in Gram-positive bacteria, whilst the PgpB and BacA/UppP proteins are present extensively in the older bacterial phyla. The presence of PgpB across a wide range of eubacteria may be due to its importance as a PGP phosphatase (Lu et al, 2011;Touzé et al, 2008a). The high level of identity for BacA/UppP (63 %) demonstrates clearly the evolutionary pressure to maintain the functionality of this protein and also raises further questions about the biological relevance of the other proteins.…”

“…Also included recently in the PTP-lipid phosphatase family is a class 3 VH1-like DUSP mitochondrial enzyme, PTPMT1, whose preferred substrate is PGP (Tonks, 2013;Xiao et al, 2011;Zhang et al, 2011). Interestingly, PGP also serves as the substrate for the bacterial enzymes, PgpA, PgpB and PgpC (Lu et al, 2011), whilst, as noted previously, PgpB also contains partial undecaprenyl pyrophosphate phosphatase activity (Touzé et al, 2008a). The substrates for PTEN, BacA/UppP and PTPMT1 are shown in Fig.…”

“…Similarly, the protein YeiU/LpxT has also been argued to catalyse the transfer of a phosphate group from the undecaprenyl pyrophosphate donor to the 1-position of lipid A to form lipid A 1-diphosphate on the periplasmic side of the inner membrane (Touzé et al, 2008b;Valvano, 2008). To date, the enzymic specificity towards undecaprenyl pyrophosphate has only been analysed for PgpB where it primarily acts upon PGP and has minimal activity towards undecaprenyl pyrophosphate (Touzé et al, 2008a). These enzymes have therefore not been compared directly with each other to assess their relative activity on undecaprenyl pyrophosphate and their physiologically relevant contributions in vivo.…”

Crystal structure of lipid phosphatase Escherichia coli phosphatidylglycerophosphate phosphatase B

“…Recently, the crystal structure of phospho-N-acetylmuramic acid-pentapeptide translocase (MraY) from Aquifex aeolicus, which catalyzes the transfer of substrates to C 55 -P, was reported (11). For this process to be sustainable, the transport intermediate, undecaprenyl pyrophosphate, needs to be dephosphorylated on the periplasmic side by a phosphatase, for example, phosphatidyl-glycero-phosphatase B (PgpB, EC 3.1.3.27) (12), which may also be involved in the biogenesis of phosphatidylglycerol from phosphatidylglycerol phosphate (13). Similar dephosphorylation mechanisms of carrier lipids (e.g., dolichyl pyrophosphates) for glycan transport have been observed in yeast (14) and mammalian cells (4), and the corresponding PAP2 phosphatases (Cwh8 and DOLPP1, respectively) have been found to be located in the endoplasmic reticulum and are essential for luminal N-glycosylation of newly synthesized proteins in eukaryotic cells.…”