Phosphoglycolate phosphatase is a metabolic proofreading enzyme essential for cellular function in Plasmodium berghei

Abstract

Edited by Ruma BanerjeePlasmodium falciparum (Pf) 4-nitrophenylphosphatase has been shown previously to be involved in vitamin B1 metabolism. Here, conducting a BLASTp search, we found that 4-nitrophenylphosphatase from Pf has significant homology with phosphoglycolate phosphatase (PGP) from mouse, human, and yeast, prompting us to reinvestigate the biochemical properties of the Plasmodium enzyme. Because the recombinant PfPGP enzyme is insoluble, we performed an extended substrate screen and extensive biochemical characterization of the recombinantly expressed and purified homolog from Plasmodium berghei (Pb), leading to the identification of 2-phosphoglycolate and 2-phospho-L-lactate as the relevant physiological substrates of PbPGP. 2-Phosphoglycolate is generated during repair of damaged DNA ends, 2-phospho-L-lactate is a product of pyruvate kinase side reaction, and both potently inhibit two key glycolytic enzymes, triosephosphate isomerase and phosphofructokinase. Hence, PGP-mediated clearance of these toxic metabolites is vital for cell survival and functioning. Our results differ significantly from those in a previous study, wherein the PfPGP enzyme has been inferred to act on 2-phospho-D-lactate and not on the L isomer. Apart from resolving the substrate specificity conflict through direct in vitro enzyme assays, we conducted PGP gene knockout studies in P. berghei, confirming that this conserved metabolic proofreading enzyme is essential in Plasmodium. In summary, our findings establish PbPGP as an essential enzyme for normal physiological function in P. berghei and suggest that drugs that spe-cifically inhibit Plasmodium PGP may hold promise for use in anti-malarial therapies.The haloacid dehalogenase superfamily (HADSF) 5 is a large family of enzymes consisting mainly of phosphatases and phosphotransferases, which are both intracellular and extracellular in nature. These enzymes are characterized by the presence of a core Rossmanoid fold and a cap domain (1, 2). Studies of HADSF members have focused on identifying their physiological substrates by screening a wide range of metabolites that include sugar phosphates, lipid phosphates, nucleotides, as well as phosphorylated amino acids and co-factors. This approach has helped us understand the physiological relevance of these enzymes in various cellular processes, such as cell wall synthesis, catabolic and anabolic pathways, salvage pathways, signaling pathways, and detoxification (3-13). Apart from dephosphorylating metabolites, HADSF members have also been known to dephosphorylate proteins, and such members are characterized by the absence of the cap domain (1, 2). A largescale study reported by Huang et al. (5) has identified a HADSF member from Salmonella enterica that catalyzes dephosphorylation of more than 100 phosphorylated substrates (5). This extended substrate specificity is a common observation in HADSF members and often leads to a confounding situation where determining the physiological substrate of such promiscuous enzymes becomes a chall...

The Metabolite Repair Enzyme Phosphoglycolate Phosphatase Regulates Central Carbon Metabolism and Fosmidomycin Sensitivity in Plasmodium falciparum

“…Interestingly, a recent study suggested that the P. berghei PGP selectively utilized 2-phospho- l -lactate over 2-phospho- d -lactate in vitro (15), raising the possibility that this enzyme acts on both enantiomers in vivo depending on their intracellular concentrations.…”

“…falciparum dephosphorylates a range of metabolites, including thiamine pyrophosphate, and its homolog in P. berghei was reported to be essential for regulating 2-phosphoglycolate and 2-phospholactate levels (15, 16). Here, we establish in P.…”

New Mammalian Glycerol-3-Phosphate Phosphatase: Role in β-Cell, Liver and Adipocyte Metabolism

“…Therefore, the possibility that 2-phosphoglycolate, 4-phosphoerythronate and 2-phospholactate serve as 'physiological' substrates for PGP/G3PP is remote, unlike glycerol-3-phosphate, which is present at above its Km concentration in cells normally, and is readily available as substrate for G3PP. However, as 4-phosphoerythronate and 2phospholactate accumulate when PGP expression is suppressed (32)(33)(34), the possibility that PGP/G3PP may act on these substrates with very low efficiency due to their extremely low intracellular concentrations, cannot be discounted. Even though purified recombinant mouse PGP was found to show high catalytic efficiency with 2-phosphoglycolate, 4phosphoerythronate, and 2-phospholactate, and relatively lower activity with Gro3P (22,32), it also shows very high activity with the non-physiological substrate p-nitrophenol phosphate (30).…”