Publications

• Role of 2-phosphoglycolate phosphatase of Escherichia coli in metabolism of the 2-phosphoglycolate formed in DNA repair.

  Pellicer M.T., Nunez M.F., Aguilar J., Badia J., Baldoma L.

  The enzyme 2-phosphoglycolate phosphatase from Escherichia coli, encoded by the gph gene, was purified and characterized. The enzyme was highly specific for 2-phosphoglycolate and showed good catalytic efficiency (k(cat)/K(m)), which enabled the conversion of this substrate even at low intracellul ... >> More

  The enzyme 2-phosphoglycolate phosphatase from Escherichia coli, encoded by the gph gene, was purified and characterized. The enzyme was highly specific for 2-phosphoglycolate and showed good catalytic efficiency (k(cat)/K(m)), which enabled the conversion of this substrate even at low intracellular concentrations. A comparison of the structural and functional features of this enzyme with those of 2-phosphoglycolate phosphatases of different origins showed a high similarity of the sequences, implying the use of the same catalytic mechanism. Western blot analysis revealed constitutive expression of the gph gene, regardless of the carbon source used, growth stage, or oxidative stress conditions. We showed that this housekeeping enzyme is involved in the dissimilation of the intracellular 2-phosphoglycolate formed in the DNA repair of 3'-phosphoglycolate ends. DNA strand breaks of this kind are caused by agents such as the radiomimetic compound bleomycin. The differential response between a 2-phosphoglycolate phosphatase-deficient mutant and its parental strain after treatment with bleomycin allowed us to connect the intracellular formation of 2-phosphoglycolate with the production of glycolate, which is subsequently incorporated into general metabolism. We thus provide evidence for a salvage function of 2-phosphoglycolate phosphatase in the metabolism of a two-carbon compound generated by the cellular DNA repair machinery. << Less

  J. Bacteriol. 185:5815-5821(2003) [PubMed] [EuropePMC]

• Genome-wide analysis of substrate specificities of the Escherichia coli haloacid dehalogenase-like phosphatase family.

  Kuznetsova E., Proudfoot M., Gonzalez C.F., Brown G., Omelchenko M.V., Borozan I., Carmel L., Wolf Y.I., Mori H., Savchenko A.V., Arrowsmith C.H., Koonin E.V., Edwards A.M., Yakunin A.F.

  Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized ... >> More

  Haloacid dehalogenase (HAD)-like hydrolases are a vast superfamily of largely uncharacterized enzymes, with a few members shown to possess phosphatase, beta-phosphoglucomutase, phosphonatase, and dehalogenase activities. Using a representative set of 80 phosphorylated substrates, we characterized the substrate specificities of 23 soluble HADs encoded in the Escherichia coli genome. We identified small molecule phosphatase activity in 21 HADs and beta-phosphoglucomutase activity in one protein. The E. coli HAD phosphatases show high catalytic efficiency and affinity to a wide range of phosphorylated metabolites that are intermediates of various metabolic reactions. Rather than following the classical "one enzyme-one substrate" model, most of the E. coli HADs show remarkably broad and overlapping substrate spectra. At least 12 reactions catalyzed by HADs currently have no EC numbers assigned in Enzyme Nomenclature. Surprisingly, most HADs hydrolyzed small phosphodonors (acetyl phosphate, carbamoyl phosphate, and phosphoramidate), which also serve as substrates for autophosphorylation of the receiver domains of the two-component signal transduction systems. The physiological relevance of the phosphatase activity with the preferred substrate was validated in vivo for one of the HADs, YniC. Many of the secondary activities of HADs might have no immediate physiological function but could comprise a reservoir for evolution of novel phosphatases. << Less

  J. Biol. Chem. 281:36149-36161(2006) [PubMed] [EuropePMC]

  This publication is cited by 7 other entries.

• Phosphoglycolate phosphatase. Purification and properties.

  Christeller J.T., Tolbert N.E.

  Phosphoglycolate phosphatase (EC 3.1.3.18) was purified 1500-fold from field-grown tobacco leaves by acetone fractionation, DEAE-cellulose and molecular sieve chromatography, and preparative polyacrylamide gel electrophoresis. Preparations were judged 90 to 95% homogeneous by chromatography on DEA ... >> More

  Phosphoglycolate phosphatase (EC 3.1.3.18) was purified 1500-fold from field-grown tobacco leaves by acetone fractionation, DEAE-cellulose and molecular sieve chromatography, and preparative polyacrylamide gel electrophoresis. Preparations were judged 90 to 95% homogeneous by chromatography on DEAE-cellulose, polyacrylamide gel electrophoresis, and by isoelectric focusing. The highest specific activity obtained was 468 mumol of phosphate released/min/mg of protein. The native protein has a molecular weight of 80,500 by Ferguson plot analysis and 86,300 by sedimentation velocity on sucrose density gradients. Sodium dodecyl sulfate-polyacrylamide gels gave a molecular weight of 20,700, indicating the P-glycolate phosphatase is a tetramer with identical or near identical subunits. The enzyme, freshly purified or in crude homogenates, had a pI of 3.8 to 3.9 pH units by isoelectric focusing. Phosphosphoglycolate phosphatase from spinach leaves has a molecular weight of 93,000 and, unlike the enzyme from tobacco leaves, it is extremely unstable after DEAE-cellulose chromatography and is inactivated by lipase (EC 3.1.1.3). The phosphatase from both plants was stabilized by the addition of citrate or isocitrate in the buffers. Ribose 5-phosphate is a competitive inhibitor of phosphoglycolate phosphatase at physiological concentration, while other phosphate esters of the photosynthetic carbon cycle were without effect. << Less

  J Biol Chem 253:1780-1785(1978) [PubMed] [EuropePMC]

• Identification of the photorespiratory 2-phosphoglycolate phosphatase, PGLP1, in Arabidopsis.

  Schwarte S., Bauwe H.

  The chloroplastidal enzyme 2-phosphoglycolate phosphatase (PGLP), PGLP1, catalyzes the first reaction of the photorespiratory C(2) cycle, a major pathway of plant primary metabolism. Thirteen potential PGLP genes are annotated in the Arabidopsis (Arabidopsis thaliana) genome; however, none of thes ... >> More

  The chloroplastidal enzyme 2-phosphoglycolate phosphatase (PGLP), PGLP1, catalyzes the first reaction of the photorespiratory C(2) cycle, a major pathway of plant primary metabolism. Thirteen potential PGLP genes are annotated in the Arabidopsis (Arabidopsis thaliana) genome; however, none of these genes has been functionally characterized, and the gene encoding the photorespiratory PGLP is not known. Here, we report on the identification of the PGLP1 gene in a higher plant and provide functional evidence for a second, nonphotorespiratory PGLP, PGLP2. Two candidate genes, At5g36700 (AtPGLP1) and At5g47760 (AtPGLP2), were selected by sequence similarity to known PGLPs from microorganisms. The two encoded proteins were overexpressed in Escherichia coli and both show PGLP activity. T-DNA knockout of one of these genes, At5g36700, results in very low leaf PGLP activity. The mutant is unviable in normal air but grows well in air enriched with 0.9% CO(2). In contrast, deletion of At5g47760 does not result in a visible phenotype, and leaf PGLP activity is unaltered. Sequencing of genomic DNA from another PGLP-deficient mutant revealed a combined missense and missplicing point mutation in At5g36700. These combined data establish At5g36700 as the gene encoding the photorespiratory PGLP, PGLP1. << Less

  Plant Physiol. 144:1580-1586(2007) [PubMed] [EuropePMC]

• Structure of a haloacid dehalogenase superfamily phosphatase PH1421 from Pyrococcus horikoshii OT3: oligomeric state and thermoadaptation mechanism.

  Yamamoto H., Takio K., Sugahara M., Kunishima N.

  PH1421 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is a hypothetical protein belonging to the haloacid dehalogenase (HAD) superfamily. To gain insight into its biological function and thermostabilization mechanism, the crystal structure of PH1421 has been determined at 1.6 A reso ... >> More

  PH1421 from the hyperthermophilic archaeon Pyrococcus horikoshii OT3 is a hypothetical protein belonging to the haloacid dehalogenase (HAD) superfamily. To gain insight into its biological function and thermostabilization mechanism, the crystal structure of PH1421 has been determined at 1.6 A resolution. The crystallographic asymmetric unit contains a homodimer. The monomeric protomer is composed of two distinct domains, a small cap domain and a large core domain, which agrees well with the typical domain organization of HAD subfamily II. Based on structure-based amino-acid sequence alignment and enzymatic analysis, PH1421 is suggested to be a magnesium-dependent phosphatase that is similar to the dimeric HAD phosphatase TA0175 from the mesothermophilic archaeon Thermoplasma acidophilum. Further comparison between the crystal structures of PH1421 and TA0175 revealed a marked structural similarity in the interprotomer dimer association. The common dimer interface with interprotomer twofold symmetry is characterized by a well conserved hydrophobic core consisting of the beta1-alpha1 loop and helices alpha1 and alpha2 of the core domain and additional contacts including the beta7-beta8 loop of the cap domain, which constitutes part of the putative active site of the enzyme. Several factors that potentially contribute to the higher thermal stability of PH1421 were identified: (i) an increase in intraprotomer hydrophobic interactions, (ii) a decrease in denaturation entropy from amino-acid composition and (iii) an increased number of intraprotomer ion pairs. These results suggest that the PH1421 protomer itself has an intrinsically higher thermal stability when compared with the mesothermophilic orthologue TA0175. << Less

  Acta Crystallogr. D 64:1068-1077(2008) [PubMed] [EuropePMC]

• Structure- and function-based characterization of a new phosphoglycolate phosphatase from Thermoplasma acidophilum.

  Kim Y., Yakunin A.F., Kuznetsova E., Xu X., Pennycooke M., Gu J., Cheung F., Proudfoot M., Arrowsmith C.H., Joachimiak A., Edwards A.M., Christendat D.

  The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Using a combination of amino acid sequence analysis, three-dimensional crystal structure infor ... >> More

  The protein TA0175 has a large number of sequence homologues, most of which are annotated as unknown and a few as belonging to the haloacid dehalogenase superfamily, but has no known biological function. Using a combination of amino acid sequence analysis, three-dimensional crystal structure information, and kinetic analysis, we have characterized TA0175 as phosphoglycolate phosphatase from Thermoplasma acidophilum. The crystal structure of TA0175 revealed two distinct domains, a larger core domain and a smaller cap domain. The large domain is composed of a centrally located five-stranded parallel beta-sheet with strand order S10, S9, S8, S1, S2 and a small beta-hairpin, strands S3 and S4. This central sheet is flanked by a set of three alpha-helices on one side and two helices on the other. The smaller domain is composed of an open faced beta-sandwich represented by three antiparallel beta-strands, S5, S6, and S7, flanked by two oppositely oriented alpha-helices, H3 and H4. The topology of the large domain is conserved; however, structural variation is observed in the smaller domain among the different functional classes of the haloacid dehalogenase superfamily. Enzymatic assays on TA0175 revealed that this enzyme catalyzed the dephosphorylation of phosphoglycolate in vitro with similar kinetic properties seen for eukaryotic phosphoglycolate phosphatase. Activation by divalent cations, especially Mg2+, and competitive inhibition behavior with Cl-ions are similar between TA0175 and phosphoglycolate phosphatase. The experimental evidence presented for TA0175 is indicative of phosphoglycolate phosphatase. << Less

  J. Biol. Chem. 279:517-526(2004) [PubMed] [EuropePMC]

• Phosphoglycolate phosphatase is a metabolic proofreading enzyme essential for cellular function in <i>Plasmodium berghei</i>.

  Kempaiah Nagappa L., Satha P., Govindaraju T., Balaram H.

  <i>Plasmodium falciparum</i> (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 yea ... >> More

  <i>Plasmodium falciparum</i> (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 <i>Plasmodium</i> 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 <i>Plasmodium berghei</i> (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 <i>in vitro</i> enzyme assays, we conducted PGP gene knockout studies in <i>P. berghei</i>, confirming that this conserved metabolic proofreading enzyme is essential in <i>Plasmodium</i> In summary, our findings establish PbPGP as an essential enzyme for normal physiological function in <i>P. berghei</i> and suggest that drugs that specifically inhibit <i>Plasmodium</i> PGP may hold promise for use in anti-malarial therapies. << Less

  J Biol Chem 294:4997-5007(2019) [PubMed] [EuropePMC]