Abstract
We provided genetic and biochemical evidence that supported the conclusion that the product of pgpB gene of Escherichia coli exhibited diacylglycerol pyrophosphate (DGPP) phosphatase activity. DGPP phosphatase activity was absent in pgpB mutant cells and was expressed at high levels in cells carrying the wild-type pgpB gene on a runaway replication plasmid. The pgpB mutant has been primarily characterized by a defect in phosphatidate (PA) phosphatase activity and also exhibits defects in lyso-PA phosphatase and phosphatidylglycerophosphate phosphatase activities. The defective PA phosphatase in the pgpB mutant was shown to be a Mg2+-independent PA phosphatase activity of the DGPP phosphatase enzyme. We characterized DGPP phosphatase activity in membranes from cells overproducing the pgpB gene product. DGPP phosphatase catalyzed the dephosphorylation of the beta phosphate of DGPP to form PA followed by the dephosphorylation of PA to form diacylglycerol. The specificity constant (Vmax/Km) for DGPP was 9.3-fold greater than that for PA. The pH optimum for the DGPP phosphatase reaction was 6. 5. Activity was independent of a divalent cation requirement, was potently inhibited by Mn2+ ions, and was insensitive to inhibition by N-ethylmaleimide. Pure DGPP phosphatase from Saccharomyces cerevisiae was shown to be similar to the E. coli DGPP phosphatase in its ability to utilize lyso-PA and phosphatidylglycerophosphate as substrates in vitro.
Highlights
DGPP1 is a novel phospholipid that was first identified from the plant Catharanthus roseus (1)
We provided genetic and biochemical evidence that supported the conclusion that the product of pgpB gene of Escherichia coli exhibited diacylglycerol pyrophosphate (DGPP) phosphatase activity
PA Phosphatase and DGPP Phosphatase Activities in pgpB Mutant Cells and in Cells Overexpressing the pgpB Gene Product—The original E. coli pgpB mutant was isolated in a biochemical screen that was designed to isolate cells defective in PGP phosphatase activity (23)
Summary
DGPP1 is a novel phospholipid that was first identified from the plant Catharanthus roseus (1). The PA phosphatase activity of the DGPP phosphatase enzyme does not have a Mg2ϩ ion requirement and is insensitive to NEM (4). The conventional form of the enzyme associates with the endoplasmic reticulum, has a Mg2ϩ ion requirement, is inhibited by NEM, and is responsible for the synthesis of phospholipids and triacylglycerols (21). The other form of the enzyme is associated with the plasma membrane, does not have a Mg2ϩ ion requirement, is insensitive to NEM, and is thought to be involved in lipid signaling pathways (21). The PA phosphatase activity in pgpB mutants has been measured using the assay conditions described for the conventional Mg2ϩ-dependent PA phosphatase enzyme (23– 25) These assay conditions would not reveal the existence of a Mg2ϩ-independent PA phosphatase activity such as that displayed by the DGPP phosphatase enzyme. We tested the hypothesis that E. coli possessed a Mg2ϩ-independent PA phosphatase activity of a DGPP phos-
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