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3-Phosphoglycerate

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Nuno Empadinhas – One of the best experts on this subject based on the ideXlab platform.

Milton S. Da Costa – One of the best experts on this subject based on the ideXlab platform.

  • Mycobacterium tuberculosis Rv2419c, the missing glucosyl-3-Phosphoglycerate phosphatase for the second step in methylglucose lipopolysaccharide biosynthesis.
    Scientific reports, 2011
    Co-Authors: Vitor Mendes, Ana Maranha, Susana Alarico, Milton S. Da Costa, Nuno Empadinhas
    Abstract:

    Mycobacteria synthesize intracellular methylglucose lipopolysaccharides (MGLP) proposed to regulate fatty acid synthesis. Although their structures have been elucidated, the identity of most biosynthetic genes remains unknown. The first step in MGLP biosynthesis is catalyzed by a glucosyl-3-Phosphoglycerate synthase (GpgS, Rv1208 in Mycobacterium tuberculosis H37Rv). However, a typical glucosyl-3-Phosphoglycerate phosphatase (GpgP, EC3.1.3.70) for dephosphorylation of glucosyl-3-Phosphoglycerate to glucosylglycerate, was absent from mycobacterial genomes. We purified the native GpgP from Mycobacterium vanbaalenii and identified the corresponding gene deduced from amino acid sequences by mass spectrometry. The M. tuberculosis ortholog (Rv2419c), annotated as a putative phosphoglycerate mutase (PGM, EC5.4.2.1), was expressed and functionally characterized as a new GpgP. Regardless of the high specificity for glucosyl-3-Phosphoglycerate, the mycobacterial GpgP is not a sequence homolog of known isofunctional GpgPs. The assignment of a new function in M. tuberculosis genome expands our understanding of this organism’s genetic repertoire and of the early events in MGLP biosynthesis.

  • Functional and structural characterization of a novel mannosyl-3-Phosphoglycerate synthase from Rubrobacter xylanophilus reveals its dual substrate specificity.
    Molecular microbiology, 2010
    Co-Authors: Nuno Empadinhas, Pedro Pereira, Luciana Albuquerque, Joana Costa, Bebiana Sá-moura, Alexandra T. Marques, Sandra Macedo-ribeiro, Milton S. Da Costa
    Abstract:

    Rubrobacter xylanophilus is the only actinobacterium known to accumulate the organic solute mannosylglycerate (MG); moreover, the accumulation of MG is constitutive. The key enzyme for MG synthesis, catalysing the conversion of GDP-mannose (GDP-Man) and D-3-Phosphoglycerate (3-PGA) into the phosphorylated intermediate mannosyl-3-Phosphoglycerate and GDP, was purified from R. xylanophilus cell extracts and the corresponding gene was expressed in E. coli. Despite the related solute glucosylglycerate (GG) having never been detected in R. xylanophilus, the cell extracts and the pure recombinant mannosyl-3-Phosphoglycerate synthase (MpgS) could also synthesize glucosyl-3-Phosphoglycerate (GPG), the precursor of GG, in agreement with the higher homology of the novel MpgS towards GPG-synthesizing mycobacterial glucosyl-3-Phosphoglycerate synthases (GpgS) than towards MpgSs from hyper/thermophiles, known to accumulate MG under salt or thermal stress. To understand the specificity and substrate ambiguity of this novel enzyme, we determined the crystal structure of the unliganded MpgS and of its complexes with the nucleotide and sugar donors, at 2.2, 2.8 and 2.5 A resolution respectively. The first three-dimensional structures of a protein from this extremely gamma-radiation-resistant thermophile here reported show that MpgS (GT81 family) contains a GT-A like fold and clearly explain its nucleotide and sugar-donor specificity. In the GDP-Man complex, a flexible loop ((254) RQNRHQ(259) ), located close to the active site moves towards the incoming sugar moiety, providing the ligands for both magnesium ion co-ordination and sugar binding. A triple mutant of R. xylanophilus MpgS, mimicking the (206) PLAGE(210) loop stabilizing hydrogen bond network observed for mycobacterial GpgSs, reduces significantly the affinity to GDP-Man, implicating this loop in the sugar-donor discrimination.

  • Identification of the mycobacterial glucosyl-3-Phosphoglycerate synthase
    FEMS microbiology letters, 2008
    Co-Authors: Nuno Empadinhas, Vitor Mendes, Luciana Albuquerque, Sandra Macedo-ribeiro, Milton S. Da Costa
    Abstract:

    Abstract Mycobacteria synthesize unique polysaccharides that regulate fatty acid synthesis, namely the methylglucose lipopolysaccharide (MGLP) and the methylmannose polysaccharide. Glucosyl-(1–>2)-glycerate is found at the reducing end of MGLP. The mycobacterial gene encoding a glucosyl-3-Phosphoglycerate synthase (GpgS), primarily found in actinobacteria and sharing very low amino acid identity with known homo-functional GpgSs, has been identified. This gene has been annotated as an inverting family 2 glycosyltransferase of unknown function. The gpgS genes from the fast-growing Mycobacterium smegmatis strain 1102 and from the slow-growing Mycobacterium bovis BCG in Escherichia coli were expressed, and the recombinant enzymes were purified and characterized. The substrates for optimal activity were UDP-glucose and d-3-Phosphoglycerate but ADP-glucose was also an efficient donor. The enzymes had maximal activity around 45 degrees C, pH 8.0, and were strictly dependent on Mg(2+). In Mycobacterium tuberculosis H37Rv, the gene encoding GpgS (Rv1208) is identical to the homologue in Mycobacterium bovis BCG and was considered to be essential for growth. It is shown that these genes encode retaining family 81 glycosyltransferases regardless of the low amino acid identity with other known enzymes of this family.

Natalia K. Nagradova – One of the best experts on this subject based on the ideXlab platform.

Leonid E. Fridlyand – One of the best experts on this subject based on the ideXlab platform.

Natalia A. Khoroshilova – One of the best experts on this subject based on the ideXlab platform.

  • Interaction between d-glyceraldehyde-3-phosphate dehydrogenase and 3-Phosphoglycerate kinase and its functional consequences
    FEBS Letters, 1992
    Co-Authors: Natalia A. Khoroshilova, Vladimir I Muronetz, Natalia K. Nagradova
    Abstract:

    Abstract E. Coli d -glyceraldehyde-3-phosphate dehydrogenase covalently bound to Sepharose was shown to form a complex with soluble E. coli 3-Phosphoglycerate kinase with a stoichiometry of 1.77±0.61 kinase molecules per tetramer of the dehydrogenase and an apparent Kd of 1.03±0.68μM (10 mM sodium phosphate, 0.15 M NaCl). No interaction was detected between E. coli d -glyceraldehyde-3-phosphate dehydrogenase and rabbit muscle 3-Phosphoglycerate kinase. The species-specificity of the bienzyme association made it possible to develop a kinetic approach to demonstrate the functionally significant interaction between E. coli d -glyceraldehyde-3-phosphate dehydrogenase and E. coli 3-Phosphoglycerate kinase, which consists of an increase in steady-state rate of the coupled reaction.