-
Microbiology (Reading, England) Apr 2012Burkholderia phymatum STM815 is a β-rhizobial strain that can effectively nodulate several species of the large legume genus Mimosa. Two Tn5-induced mutants of this...
Burkholderia phymatum STM815 is a β-rhizobial strain that can effectively nodulate several species of the large legume genus Mimosa. Two Tn5-induced mutants of this strain, KM16-22 and KM51, failed to form root nodules on Mimosa pudica, but still caused root hair deformation, which is one of the early steps of rhizobial infection. Both mutants grew well in a complex medium. However, KM16-22 could not grow on minimal medium unless a sugar and a metabolic intermediate such as pyruvate were provided, and KM51 also could not grow on minimal medium unless a sugar was added. The Tn5-interrupted genes of the mutants showed strong homologies to pgm, which encodes 2,3-biphosphoglycerate-dependent phosphoglycerate mutase (dPGM), and fbp, which encodes fructose 1,6-bisphosphatase (FBPase). Both enzymes are known to be involved in obligate steps in carbohydrate metabolism. Enzyme assays confirmed that KM16-22 and KM51 had indeed lost dPGM and FBPase activity, respectively, whilst the activities of these enzymes were expressed normally in both free-living bacteria and symbiotic bacteroids of the parental strain STM815. Both mutants recovered their enzyme activity after the introduction of wild-type pgm or fbp genes, were subsequently able to use carbohydrate as a carbon source, and were able to form root nodules on M. pudica and to fix nitrogen as efficiently as the parental strain. We conclude that the enzymes dPGM and FBPase are essential for the formation of a symbiosis with the host plant.
Topics: Bacterial Proteins; Burkholderia; Carbohydrate Metabolism; Cloning, Molecular; Fructose-Bisphosphatase; Genetic Complementation Test; Mimosa; Mutagenesis; Nitrogen Fixation; Phosphoglycerate Mutase; Plant Root Nodulation; Plant Roots; Symbiosis
PubMed: 22282515
DOI: 10.1099/mic.0.055095-0 -
Biochimica Et Biophysica Acta Aug 1996The small, monomeric, phosphoglycerate mutase (PGAM) from Schizosaccharomyces pombe has been overexpressed in a strain of Saccharomyces cerevisiae in which the gene...
The small, monomeric, phosphoglycerate mutase (PGAM) from Schizosaccharomyces pombe has been overexpressed in a strain of Saccharomyces cerevisiae in which the gene encoding PGAM has been deleted, with a yield of purified enzyme of 10-15 mg per litre cell culture. Three mutants in which histidine residues in S. pombe PGAM have been substituted by glutamine have been purified and characterised. Two mutants (H151Q and H196Q) have kinetic and structural properties very similar to wild-type enzyme, consistent with the proposed location of these (non-conserved) histidines on the surface of the enzyme. The third mutant (H163Q) involving a histidine thought to be part of the active site has greatly reduced mutase and phosphatase activities. Mass spectrometry shows that the phosphorylated form of the H163Q is several 100-times more stable towards hydrolysis than the phosphorylated form of wild-type enzyme. The H163Q mutant appears to be structurally quite distinct from wild-type enzyme. 600 MHz 1D proton NMR spectra of good quality have been obtained for wild-type enzyme and the H151Q and H196Q mutants.
Topics: Base Sequence; Circular Dichroism; Histidine; Kinetics; Magnetic Resonance Spectroscopy; Mass Spectrometry; Models, Molecular; Molecular Sequence Data; Mutagenesis, Site-Directed; Mutation; Phosphoglycerate Mutase; Recombinant Proteins; Schizosaccharomyces; Spectrometry, Fluorescence
PubMed: 8765231
DOI: 10.1016/0167-4838(96)00046-5 -
Comparative Biochemistry and... 1984Treatment of rabbit muscle phosphoglycerate mutase with trinitrobenzenesulfonate and with pyridoxal-5'-phosphate produces the concurrent loss of the three activities of...
Metabolism of glycerate-2,3-P2--VI. Lysyl-specific reagents inactivate the phosphoglycerate mutase, glycerate-2,3-P2 synthase and glycerate-2,3-P2 phosphatase activities of rabbit muscle phosphoglycerate mutase.
Treatment of rabbit muscle phosphoglycerate mutase with trinitrobenzenesulfonate and with pyridoxal-5'-phosphate produces the concurrent loss of the three activities of the enzyme: phosphoglycerate mutase, glycerate-2,3-P2 synthase and glycerate-2,3-P2 phosphatase. With both reagents complete inactivation occurs with modification of about 3 moles of lysine per mole of enzyme. Inactivated phosphoglycerate mutase is unable to form the functionally active phosphoenzyme when mixed with glycerate-2,3-P2. Substrate (glycerate-3-P) protects against pyridoxal-5'-phosphate inactivation, and offers some protection against TNBS inactivation. Cofactor (glycerate-2,3-P2) does not prevent inactivation. These results provide additional evidence of the intrinsic character of the three enzymatic activities of phosphoglycerate mutase and favour their location at the same active site. In addition, they suggest that the essential lysyl residues are located at or near the substrate binding site.
Topics: Animals; Chemical Phenomena; Chemistry; Kinetics; Lysine; Muscles; Phosphoglycerate Mutase; Phosphoric Monoester Hydrolases; Phosphotransferases; Pyridoxal Phosphate; Rabbits; Trinitrobenzenesulfonic Acid
PubMed: 6325083
DOI: 10.1016/0305-0491(84)90262-1 -
Chemistry, An Asian Journal Sep 2020Here we report the construction of an mRNA-encoded library of thioether-closed macrocyclic peptides by using an N-chloroacetyl-cyclopropane-containing exotic initiator...
Here we report the construction of an mRNA-encoded library of thioether-closed macrocyclic peptides by using an N-chloroacetyl-cyclopropane-containing exotic initiator whose structure is more constrained than the ordinary N-chloroacetyl-α-amino acid initiators. The use of such an initiator has led to a macrocycle library with significantly suppressed population of lariat-shaped species compared with the conventional libraries. We previously used a conventional library and identified a small lariat thioether-macrocycle with a tail peptide with a C-terminal free Cys whose sidechain plays an essential role in potent inhibitory activity against a parasitic model enzyme, phosphoglycerate mutase. On the other hand, the cyclopropane-containing macrocycle library has yielded a larger thioether-macrocycle lacking a free Cys residue, which exhibits potent inhibitory activity to the same enzyme with a different mode of action. This result indicates that such a cyclopropane-containing macrocycle library would allow us to access mechanistically distinct macrocycles.
Topics: Animals; Caenorhabditis elegans; Cyclopropanes; Enzyme Inhibitors; Macrocyclic Compounds; Molecular Structure; Peptide Library; Peptides; Phosphoglycerate Mutase; Sulfhydryl Compounds
PubMed: 32633882
DOI: 10.1002/asia.202000700 -
Comparative Biochemistry and... Oct 1995We have previously demonstrated that maize (Zea mays) 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAM-i) is not related to... (Comparative Study)
Comparative Study
We have previously demonstrated that maize (Zea mays) 2,3-bisphosphoglycerate-independent phosphoglycerate mutase (PGAM-i) is not related to 2,3-bisphosphoglycerate-dependent phosphoglycerate mutase. With the aid of specific anti-maize PGAM-i antibodies, we demonstrate here the presence of a closely related PGAM-i in other plants. We also describe the isolation and sequencing of a cDNA-encoding almond (Prunus amygdalus) PGAM-i that further demonstrates this relationship among plant PGAM-i. A search of the major databases for related sequences allowed us to identify some novel PGAM-i from different sources: plants (Arabidopsis thaliana, Oryza sativa and Antithamniom sp.), monera (Escherichia coli, Bacillus subtilis and Bacillus megaterium) and animals (Caenorhabditis elegans). All of these amino acid sequences share a high degree of homology with plant PGAM-i. These observations suggest that the PGAM-i from several biological kingdoms constitute a family of protein different from other proteins with related enzymatic function and arose from a common ancestral gene that has diverged throughout its evolution.
Topics: Amino Acid Sequence; Animals; Bacteria; Biological Evolution; Conserved Sequence; DNA, Complementary; Molecular Sequence Data; Phosphoglycerate Mutase; Plants; Sequence Alignment; Sequence Analysis
PubMed: 7584858
DOI: 10.1016/0305-0491(95)00076-3 -
Biochimica Et Biophysica Acta Jan 2008Histidine phosphorylation is important in prokaryotes and occurs to the extent of 6% of total phosphorylation in eukaryotes. Nevertheless phosphohistidine residues are... (Review)
Review
Histidine phosphorylation is important in prokaryotes and occurs to the extent of 6% of total phosphorylation in eukaryotes. Nevertheless phosphohistidine residues are not normally observed in proteins due to rapid hydrolysis of the phosphoryl group under acidic conditions. Many rapid processes employ phosphohistidines, including the bacterial phosphoenolpyruvate:sugar phosphotransferase system (PTS), the bacterial two-component systems and reactions catalyzed by enzymes such as nucleoside diphosphate kinase and succinyl-CoA synthetase. In the PTS, the NMR structure of the phosphohistidine moiety of the phosphohistidine-containing protein was determined but no X-ray structures of phosphohistidine forms of PTS proteins have been elucidated. There have been crystal structures of a few phosphohistidine-containing proteins determined: nucleoside diphosphate kinase, succinyl-CoA synthetase, a cofactor-dependent phosphoglycerate mutase and the protein PAE2307 from the hyperthermophilic archaeon Pyrobaculum aerophilum. A common theme for these stable phosphohistidines is the occurrence of ion-pair hydrogen bonds (salt bridges) involving the non-phosphorylated nitrogen atom of the histidine imidazole ring with an acidic amino acid side chain.
Topics: Crystallography, X-Ray; Histidine; Hydrogen Bonding; Nucleoside-Diphosphate Kinase; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphoglycerate Mutase; Phosphorylation; Succinate-CoA Ligases
PubMed: 17728195
DOI: 10.1016/j.bbapap.2007.07.008 -
Gene Feb 2001Two mouse cDNAs encoding the non-muscle-specific or brain isoform (type B, Pgam1) and the muscle-specific isoform (type M, Pgam2) of phosphoglycerate mutase (PGAM) were...
Two mouse cDNAs encoding the non-muscle-specific or brain isoform (type B, Pgam1) and the muscle-specific isoform (type M, Pgam2) of phosphoglycerate mutase (PGAM) were isolated and characterized. Pgam1 contains a 765 bp open reading frame (ORF) coding for a 254-residue protein while Pgam2 contains a 762 bp ORF coding for a 253-residue protein. The deduced proteins of mouse Pgam1 and Pgam2 are highly similar to those of human and rat (> or = 93% similarity). Northern blot analysis showed that the expression patterns of Pgam1 and Pgam2 were distinct. Pgam1 was expressed as a 2.1-kb transcript highly in brain and kidney and moderately in liver, thyroid, stomach and heart, whereas Pgam2 was expressed as a 1.0-kb transcript highly in muscle, testis and moderately in heart and lung, but was not detectable in the other six tissues examined. Transfecting the cDNA fragments containing the entire ORFs of these two cDNAs into COS7 cells for transient expression, respectively, the enzyme activities of mouse Pgam1 and Pgam2 were detected to be 2.2-2.5 times of those of COS7 cells and COS7 cells transfected with vector, proving the validity of mouse Pgam1 and Pgam2 cDNAs we report here. Pgam1 and Pgam2 were assigned to 116.16 cR from D19Mit52 and 29.57 cR from D11Mit129, respectively, by radiation hybrid method. The partial genomic sequence of Pgam2 was determined, which showed that mouse Pgam2 consisted at least three exons and two introns. In addition, a pseudogene of Pgam1, Pgam1-ps1, was identified from mouse genomic sequence.
Topics: Amino Acid Sequence; Animals; Base Sequence; Blotting, Northern; COS Cells; Chromosome Mapping; Cloning, Molecular; DNA, Complementary; DNA, Recombinant; Gene Expression Regulation, Enzymologic; Isoenzymes; Male; Mice; Molecular Sequence Data; Phosphoenolpyruvate; Phosphoglycerate Mutase; Plasmids; Pseudogenes; RNA, Messenger; Radiation Hybrid Mapping; Sequence Alignment; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Tissue Distribution
PubMed: 11250083
DOI: 10.1016/s0378-1119(00)00597-7 -
Plant Biology (Stuttgart, Germany) Jul 2019Glycolysis is a central metabolic pathway that provides energy and products of primary metabolites. 2,3-Biphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is...
Mutation of the rice TCM12 gene encoding 2,3-bisphosphoglycerate-independent phosphoglycerate mutase affects chlorophyll synthesis, photosynthesis and chloroplast development at seedling stage at low temperatures.
Glycolysis is a central metabolic pathway that provides energy and products of primary metabolites. 2,3-Biphosphoglycerate-independent phosphoglycerate mutase (iPGAM) is a key enzyme that catalyses the reversible interconversion of 3-phosphoglycerate (3-PGA) to 2-phosphoglycerate (2-PGA) in glycolysis. Low temperature is a common abiotic stress in rice production. However, the mechanism for rice iPGAM genes is not fully understood at low temperature. In this study, the rice mutant tcm12, with chlorosis, malformed chloroplasts and impaired photosynthesis, was grown at a low temperature (<20 °C) to the three-leaf stage, while the normal phenotype at 32 °C was used. Chlorophyll fluorescence analysis and transmission electron microscopy were used to examine features of the tcm12 mutant. The inheritance behaviour and function of TCM12 were then analysed thorough map-based cloning, transgenic complementation and subcellular localisation. The thermo-sensitive chlorosis phenotype was caused by a single nucleotide mutation (T→C) on the fifth exon of TCM12 (LOC_Os12g35040) encoding iPGAM, localised to both nucleus and membranes. In addition, TCM12 was constitutively expressed, and its disruption resulted in down-regulation of some genes associated with chlorophyll biosynthesis and photosynthesis at low temperatures (20 °C). This is the first report of the involvement of rice iPGAM gene in chlorophyll synthesis, photosynthesis and chloroplast development, providing new insights into the mechanisms underlying early growth of rice at low temperatures.
Topics: 2,3-Diphosphoglycerate; Chlorophyll; Chloroplasts; Cloning, Molecular; Cold Temperature; Genes, Plant; Microscopy, Electron, Transmission; Mutation; Oryza; Phosphoglycerate Mutase; Photosynthesis; Seedlings
PubMed: 30803106
DOI: 10.1111/plb.12978 -
Microvascular Research Aug 2008To elucidate the cellular localization of Phosphoglycerate mutase (PGAM1) type B in the brain and periphery, immunocytochemical studies were performed. The purified...
To elucidate the cellular localization of Phosphoglycerate mutase (PGAM1) type B in the brain and periphery, immunocytochemical studies were performed. The purified antigen used to generate the antiserum to PGAM1 was run on an SDS-PAGE gel, stained with coomassie blue, which yielded one sharp band at 29 kDa. Immunocytochemistry of formalin perfused rats revealed distinct localization of PGAM1 in the endothelium of the capillaries and arteries of the brain, liver and kidneys. Since enhanced glycogenesis is a well-known characteristic of cancer cells, it is of interest that sustained angiogenesis is a hallmark that distinguishes cancer cells from their normal counterparts. In view of the fact that PGAM1 increases in a variety of tumors, we suggest that PGAM1 may have a pathological role of vascular invasion into cancerous tissue.
Topics: Animals; Blotting, Western; Brain; Capillaries; Electrophoresis, Polyacrylamide Gel; Endothelium, Vascular; Immunohistochemistry; Kidney; Liver; Male; Microscopy, Confocal; Phosphoglycerate Mutase; Rats; Rats, Sprague-Dawley
PubMed: 18533197
DOI: 10.1016/j.mvr.2008.04.001 -
Acta Pharmacologica Sinica Dec 2017Phosphoglycerate mutase 1 (PGAM1), an important enzyme in glycolysis, is overexpressed in a number of human cancers, thus has been proposed as a promising metabolic...
Phosphoglycerate mutase 1 (PGAM1), an important enzyme in glycolysis, is overexpressed in a number of human cancers, thus has been proposed as a promising metabolic target for cancer treatments. The C-terminal portion of the available crystal structures of PGAM1 and its homologous proteins is partially disordered, as evidenced by weak electron density. In this study, we identified the conformational behavior of the C-terminal region of PGAM1 as well as its role during the catalytic cycle. Using the PONDR-FIT server, we demonstrated that the C-terminal region was intrinsically disordered. We applied the Monte Carlo (MC) method to explore the conformational space of the C-terminus and conducted a series of explicit-solvent molecular dynamics (MD) simulations, and revealed that the C-terminal region is inherently dynamic; large-scale conformational changes in the C-terminal segment led to the structural transition of PGAM1 from the closed state to the open state. Furthermore, the C-terminal segment influenced 2,3-bisphosphoglycerate (2,3-BPG) binding. The proposed swing model illustrated a critical role of the C-terminus in the catalytic cycle through the conformational changes. In conclusion, the C-terminal region induces large movements of PGAM1 from the closed state to the open state and influences cofactor binding during the catalytic cycle. This report describes the dynamic features of the C-terminal region in detail and should aid in design of novel and efficient inhibitors of PGAM1. A swing mechanism of the C-terminal region is proposed, to facilitate further studies of the catalytic mechanism and the physiological functions of its homologues.
Topics: Biocatalysis; Enzyme Inhibitors; Humans; Molecular Dynamics Simulation; Monte Carlo Method; Phosphoglycerate Mutase; Principal Component Analysis; Protein Conformation; Static Electricity
PubMed: 28748916
DOI: 10.1038/aps.2017.37