Publications

• Mycobacterium tuberculosis Rv2419c, the missing glucosyl-3-phosphoglycerate phosphatase for the second step in methylglucose lipopolysaccharide biosynthesis.

  Mendes V., Maranha A., Alarico S., da Costa M.S., Empadinhas N.

  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-phosph ... >> More

  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. << Less

  Sci. Rep. 1:177-177(2011) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Development of a mutagenesis, expression and purification system for yeast phosphoglycerate mutase. Investigation of the role of active-site His181.

  White M.F., Fothergill-Gilmore L.A.

  A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a ... >> More

  A system has been developed to allow the convenient production, expression and purification of site-directed mutants of the enzyme phosphoglycerate mutase from Saccharomyces cerevisiae. This enzyme is well characterised; both the amino acid sequence and crystal structure have been determined and a reaction mechanism has been proposed. However, the molecular basis for catalysis remains poorly understood, with only circumstantial evidence for the roles of most of the active site residues other than His8, which is phosphorylated during the reaction cycle. A vector/host expression system has been designed which allows recombinant forms of phosphoglycerate mutase to be efficiently expressed in yeast with no background wild-type activity. A simple one-column purification protocol typically yields 30 mg pure enzyme/1 l of culture. The active-site residue, His181, which is thought to be involved in proton transfer during the catalytic cycle, has been mutated to an alanine. The resultant mutant has been purified and characterised. Kinetic analysis shows a large decrease (1.6 x 10(4)) in the catalytic efficiency, and an 11-fold increase in the Km for the cofactor 2,3-bisphosphoglycerate. These observations are consistent with an integral role for His181 in the reaction mechanism of phosphoglycerate mutase, probably as a general acid or base. << Less

  Eur. J. Biochem. 207:709-714(1992) [PubMed] [EuropePMC]

• Unliganded structure of human bisphosphoglycerate mutase reveals side-chain movements induced by ligand binding.

  Patterson A., Price N.C., Nairn J.

  Erythrocyte-specific bisphosphoglycerate mutase is a trifunctional enzyme which modulates the levels of 2,3-bisphosphoglycerate (2,3-BPG) in red blood cells by virtue of its synthase and phosphatase activities. Low levels of erythrocyte 2,3-BPG increase the affinity of haemoglobin for oxygen, thus ... >> More

  Erythrocyte-specific bisphosphoglycerate mutase is a trifunctional enzyme which modulates the levels of 2,3-bisphosphoglycerate (2,3-BPG) in red blood cells by virtue of its synthase and phosphatase activities. Low levels of erythrocyte 2,3-BPG increase the affinity of haemoglobin for oxygen, thus limiting the release of oxygen into tissues. 2,3-BPG levels in stored blood decline rapidly owing to the phosphatase activity of bisphosphoglycerate mutase, which is enhanced by a fall in pH. Here, the 1.94 Å resolution X-ray structure of bisphosphoglycerate mutase is presented, focusing on the dynamic nature of key ligand-binding residues and their interaction with the inhibitor citrate. Residues at the binding pocket are complete. In addition, the movement of key residues in the presence and absence of ligand is described and alternative conformations are explored. The conformation in which the ligand citrate would bind at the substrate-binding pocket is proposed, with discussion and representations of its orientation. The characterization of bisphosphoglycerate mutase-citrate interactions will provide a framework for the design of specific inhibitors of the phosphatase activity of this enzyme, which may limit the decline of 2,3-BPG in stored blood. << Less

  Acta Crystallogr. F 66:1415-1420(2010) [PubMed] [EuropePMC]

• The two analogous phosphoglycerate mutases of Escherichia coli.

  Fraser H.I., Kvaratskhelia M., White M.F.

  The glycolytic enzyme phosphoglycerate mutase exists in two evolutionarily unrelated forms. Vertebrates have only the 2,3-bisphosphoglycerate-dependent enzyme (dPGM), whilst higher plants have only the cofactor-independent enzyme (iPGM). Certain eubacteria possess genes encoding both enzymes, and ... >> More

  The glycolytic enzyme phosphoglycerate mutase exists in two evolutionarily unrelated forms. Vertebrates have only the 2,3-bisphosphoglycerate-dependent enzyme (dPGM), whilst higher plants have only the cofactor-independent enzyme (iPGM). Certain eubacteria possess genes encoding both enzymes, and their respective metabolic roles and activities are unclear. We have over-expressed, purified and characterised the two PGMs of Escherichia coli. Both are expressed at high levels, but dPGM has a 10-fold higher specific activity than iPGM. Differential inhibition by vanadate was observed. The presence of an integral manganese ion in iPGM was confirmed by EPR spectroscopy. << Less

  FEBS Lett. 455:344-348(1999) [PubMed] [EuropePMC]