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Mark J Jedrzejas - One of the best experts on this subject based on the ideXlab platform.
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a cofactor dependent Phosphoglycerate Mutase homolog from bacillus stearothermophilus is actually a broad specificity phosphatase
Protein Science, 2001Co-Authors: Daniel J Rigden, Mark J Jedrzejas, Peter Setlow, Irina Bagyan, Ejvis LamaniAbstract:The distribution of Phosphoglycerate Mutase (PGM) activity in bacteria is complex, with some organisms possessing both a cofactor-dependent and a cofactor-independent PGM and others having only one of these enzymes. Although Bacillus species contain only a cofactor-independent PGM, genes homologous to those encoding cofactor-dependent PGMs have been detected in this group of bacteria, but in at least one case the encoded protein lacks significant PGM activity. Here we apply sequence analysis, molecular modeling, and enzymatic assays to the cofactor-dependent PGM homologs from B. stearothermophilus and B. subtilis, and show that these enzymes are phosphatases with broad substrate specificity. Homologs from other gram-positive bacteria are also likely to possess phosphatase activity. These studies clearly show that the exploration of genomic sequences through three-dimensional modeling is capable of producing useful predictions regarding function. However, significant methodological improvements will be needed before such analysis can be carried out automatically.
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mechanism of catalysis of the cofactor independent Phosphoglycerate Mutase from bacillus stearothermophilus crystal structure of the complex with 2 Phosphoglycerate
Journal of Biological Chemistry, 2000Co-Authors: Mark J Jedrzejas, Monica Chander, Peter Setlow, Gunasekaran KrishnasamyAbstract:The structure of the complex between the 2, 3-diPhosphoglycerate-independent Phosphoglycerate Mutase (iPGM) from Bacillus stearothermophilus and its 3-Phosphoglycerate substrate has recently been solved, and analysis of this structure allowed formulation of a mechanism for iPGM catalysis. In order to obtain further evidence for this mechanism, we have solved the structure of this iPGM complexed with 2-Phosphoglycerate and two Mn(2+) ions at 1. 7-A resolution. The structure consists of two different domains connected by two loops and interacting through a network of hydrogen bonds. This structure is consistent with the proposed mechanism for iPGM catalysis, with the two main steps in catalysis being a phosphatase reaction removing the phosphate from 2- or 3-Phosphoglycerate, generating an enzyme-bound phosphoserine intermediate, followed by a phosphotransferase reaction as the phosphate is transferred from the enzyme back to the glycerate moiety. The structure also allowed the assignment of the function of the two domains of the enzyme, one of which participates in the phosphatase reaction and formation of the phosphoserine enzyme intermediate, with the other involved in the phosphotransferase reaction regenerating Phosphoglycerate. Significant structural similarity has also been found between the active site of the iPGM domain catalyzing the phosphatase reaction and Escherichia coli alkaline phosphatase.
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structure and mechanism of action of a novel Phosphoglycerate Mutase from bacillus stearothermophilus
The EMBO Journal, 2000Co-Authors: Mark J Jedrzejas, Monica Chander, Peter Setlow, Gunasekaran KrishnasamyAbstract:Bacillus stearothermophilus Phosphoglycerate Mutase (PGM), which interconverts 2- and 3-phosphoglyceric acid (PGA), does not require 2,3-diphosphoglyceric acid for activity. However, this enzyme does have an absolute and specific requirement for Mn(2+) ions for catalysis. Here we report the crystal structure of this enzyme complexed with 3PGA and manganese ions to 1.9 A resolution; this is the first crystal structure of a diPhosphoglycerate-independent PGM to be determined. This information, plus the location of the two bound Mn(2+) ions and the 3PGA have allowed formulation of a possible catalytic mechanism for this PGM. In this mechanism Mn(2+) ions facilitate the transfer of the substrate's phosphate group to Ser62 to form a phosphoserine intermediate. In the subsequent phosphotransferase part of the reaction, the phosphate group is transferred from Ser62 to the O2 or O3 positions of the reoriented glycerate to yield the PGA product. Site-directed mutagenesis studies were used to confirm our mechanism and the involvement of specific enzyme residues in Mn(2+) binding and catalysis.
Dariusz Rakus - One of the best experts on this subject based on the ideXlab platform.
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insulin igf1 pi3k dependent nucleolar localization of a glycolytic enzyme Phosphoglycerate Mutase 2 is necessary for proper structure of nucleolus and rna synthesis
Oncotarget, 2015Co-Authors: Agnieszka Gizak, Marcin Grenda, Piotr Mamczur, Janusz Wisniewski, Filip Sucharski, Jerzy Silberring, James A Mccubrey, Jacek R Wisniewski, Dariusz RakusAbstract:// Agnieszka Gizak 1 , Marcin Grenda 1 , Piotr Mamczur 1 , Janusz Wisniewski 1 , Filip Sucharski 2 , Jerzy Silberring 2 , James A. McCubrey 3 , Jacek R. Wisniewski 4 and Dariusz Rakus 1 1 Department of Animal Molecular Physiology, Wroclaw University, Cybulskiego, Wroclaw, Poland 2 Department of Biochemistry and Neurobiology, Faculty of Materials Science and Ceramics, AGH University of Science and Technology, al. Mickiewicza, Krakow, Poland 3 Department of Microbiology and Immunology, Brody School of Medicine at East Carolina University Greenville, NC, USA 4 Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max-Planck-Institute of Biochemistry, Am Klopferspitz, Martinsried, Germany Correspondence to: Agnieszka Gizak, email: // Keywords : squamous cell carcinoma, PGAM2, rRNA, ribosome assembly, multifunctional enzyme Received : February 25, 2015 Accepted : April 30, 2015 Published : May 08, 2015 Abstract Phosphoglycerate Mutase (PGAM), a conserved, glycolytic enzyme has been found in nucleoli of cancer cells. Here, we present evidence that accumulation of PGAM in the nucleolus is a universal phenomenon concerning not only neoplastically transformed but also non-malignant cells. Nucleolar localization of the enzyme is dependent on the presence of the PGAM2 (muscle) subunit and is regulated by insulin/IGF-1–PI3K signaling pathway as well as drugs influencing ribosomal biogenesis. We document that PGAM interacts with several 40S and 60S ribosomal proteins and that silencing of PGAM2 expression results in disturbance of nucleolar structure, inhibition of RNA synthesis and decrease of the mitotic index of squamous cell carcinoma cells. We conclude that presence of PGAM in the nucleolus is a prerequisite for synthesis and initial assembly of new pre-ribosome subunits.
Peter Setlow - One of the best experts on this subject based on the ideXlab platform.
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a cofactor dependent Phosphoglycerate Mutase homolog from bacillus stearothermophilus is actually a broad specificity phosphatase
Protein Science, 2001Co-Authors: Daniel J Rigden, Mark J Jedrzejas, Peter Setlow, Irina Bagyan, Ejvis LamaniAbstract:The distribution of Phosphoglycerate Mutase (PGM) activity in bacteria is complex, with some organisms possessing both a cofactor-dependent and a cofactor-independent PGM and others having only one of these enzymes. Although Bacillus species contain only a cofactor-independent PGM, genes homologous to those encoding cofactor-dependent PGMs have been detected in this group of bacteria, but in at least one case the encoded protein lacks significant PGM activity. Here we apply sequence analysis, molecular modeling, and enzymatic assays to the cofactor-dependent PGM homologs from B. stearothermophilus and B. subtilis, and show that these enzymes are phosphatases with broad substrate specificity. Homologs from other gram-positive bacteria are also likely to possess phosphatase activity. These studies clearly show that the exploration of genomic sequences through three-dimensional modeling is capable of producing useful predictions regarding function. However, significant methodological improvements will be needed before such analysis can be carried out automatically.
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mechanism of catalysis of the cofactor independent Phosphoglycerate Mutase from bacillus stearothermophilus crystal structure of the complex with 2 Phosphoglycerate
Journal of Biological Chemistry, 2000Co-Authors: Mark J Jedrzejas, Monica Chander, Peter Setlow, Gunasekaran KrishnasamyAbstract:The structure of the complex between the 2, 3-diPhosphoglycerate-independent Phosphoglycerate Mutase (iPGM) from Bacillus stearothermophilus and its 3-Phosphoglycerate substrate has recently been solved, and analysis of this structure allowed formulation of a mechanism for iPGM catalysis. In order to obtain further evidence for this mechanism, we have solved the structure of this iPGM complexed with 2-Phosphoglycerate and two Mn(2+) ions at 1. 7-A resolution. The structure consists of two different domains connected by two loops and interacting through a network of hydrogen bonds. This structure is consistent with the proposed mechanism for iPGM catalysis, with the two main steps in catalysis being a phosphatase reaction removing the phosphate from 2- or 3-Phosphoglycerate, generating an enzyme-bound phosphoserine intermediate, followed by a phosphotransferase reaction as the phosphate is transferred from the enzyme back to the glycerate moiety. The structure also allowed the assignment of the function of the two domains of the enzyme, one of which participates in the phosphatase reaction and formation of the phosphoserine enzyme intermediate, with the other involved in the phosphotransferase reaction regenerating Phosphoglycerate. Significant structural similarity has also been found between the active site of the iPGM domain catalyzing the phosphatase reaction and Escherichia coli alkaline phosphatase.
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structure and mechanism of action of a novel Phosphoglycerate Mutase from bacillus stearothermophilus
The EMBO Journal, 2000Co-Authors: Mark J Jedrzejas, Monica Chander, Peter Setlow, Gunasekaran KrishnasamyAbstract:Bacillus stearothermophilus Phosphoglycerate Mutase (PGM), which interconverts 2- and 3-phosphoglyceric acid (PGA), does not require 2,3-diphosphoglyceric acid for activity. However, this enzyme does have an absolute and specific requirement for Mn(2+) ions for catalysis. Here we report the crystal structure of this enzyme complexed with 3PGA and manganese ions to 1.9 A resolution; this is the first crystal structure of a diPhosphoglycerate-independent PGM to be determined. This information, plus the location of the two bound Mn(2+) ions and the 3PGA have allowed formulation of a possible catalytic mechanism for this PGM. In this mechanism Mn(2+) ions facilitate the transfer of the substrate's phosphate group to Ser62 to form a phosphoserine intermediate. In the subsequent phosphotransferase part of the reaction, the phosphate group is transferred from Ser62 to the O2 or O3 positions of the reoriented glycerate to yield the PGA product. Site-directed mutagenesis studies were used to confirm our mechanism and the involvement of specific enzyme residues in Mn(2+) binding and catalysis.
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cloning and nucleotide sequences of the genes encoding triose phosphate isomerase Phosphoglycerate Mutase and enolase from bacillus subtilis
Journal of Bacteriology, 1994Co-Authors: Marco Antonio Leyvavazquez, Peter SetlowAbstract:The Bacillus subtilis genes tpi, pgm, and eno, encoding triose phosphate isomerase, Phosphoglycerate Mutase (PGM), and enolase, respectively, have been cloned and sequenced. These genes are the last three in a large putative operon coding for glycolytic enzymes; the operon includes pgk (coding for Phosphoglycerate kinase) followed by tpi, pgm, and eno. The triose phosphate isomerase and enolase from B. subtilis are extremely similar to those from all other species, both eukaryotic and prokaryotic. However, B. subtilis PGM bears no resemblance to mammalian, fungal, or gram-negative bacterial PGMs, which are dependent on 2,3-diPhosphoglycerate (DPG) for activity. Instead, B. subtilis PGM, which is DPG independent, is very similar to a DPG-independent PGM from a plant species but differs from the latter in the absolute requirement of B. subtilis PGM for Mn2+. The cloned pgm gene has been used to direct up to 25-fold overexpression of PGM in Escherichia coli; this should facilitate purification of large amounts of this novel Mn(2+)-dependent enzyme. Inactivation of pgm plus eno in B. subtilis resulted in extremely slow growth either on plates or in liquid, but growth of these mutants was enhanced by supplementation of media with malate. However, these mutants were asporogenous with or without malate supplementation.
Jian Ding - One of the best experts on this subject based on the ideXlab platform.
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Phosphoglycerate Mutase 1 promotes cancer cell migration independent of its metabolic activity
Oncogene, 2017Co-Authors: Di Zhang, Shuai Tang, Nan Jin, Wei Sun, Bo Liu, Zuoquan Xie, Xinying Yang, H Han, Dongying Chen, Jian DingAbstract:Phosphoglycerate Mutase 1 (PGAM1) is a glycolytic enzyme that coordinates glycolysis and biosynthesis to promote cancer growth via its metabolic activity. Here, we report the discovery of a non-metabolic function of PGAM1 in promoting cancer metastasis. A proteomic study identified α-smooth muscle actin (ACTA2) as a PGAM1-associated protein. PGAM1 modulated actin filaments assembly, cell motility and cancer cell migration via directly interacting with ACTA2, which was independent of its metabolic activity. The enzymatically inactive H186R mutant retained its association with ACTA2, whereas 201-210 amino acids deleted PGAM1 mutant lost the interaction with ACTA2 regardless of intact metabolic activity. Importantly, PGAM1 knockdown decreased metastatic potential of breast cancer cells in vivo and PGAM1 and ACTA2 were jointly associated with the prognosis of breast cancer patients. Together, this study provided the first evidence revealing a non-metabolic function of PGAM1 in promoting cell migration, and gained new insights into the role of PGAM1 in cancer progression.
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Phosphoglycerate Mutase 1 regulates dntp pool and promotes homologous recombination repair in cancer cells
Journal of Cell Biology, 2017Co-Authors: Wenyi Sun, Nan Jin, Zuoquan Xie, Jian Ding, Meiyu Geng, Jie Zhong, Mingrui Zhu, Minjia Tan, Shuhai LinAbstract:Glycolytic enzymes are known to play pivotal roles in cancer cell survival, yet their molecular mechanisms remain poorly understood. Phosphoglycerate Mutase 1 (PGAM1) is an important glycolytic enzyme that coordinates glycolysis, pentose phosphate pathway, and serine biosynthesis in cancer cells. Herein, we report that PGAM1 is required for homologous recombination (HR) repair of DNA double-strand breaks (DSBs) caused by DNA-damaging agents. Mechanistically, PGAM1 facilitates DSB end resection by regulating the stability of CTBP-interacting protein (CtIP). Knockdown of PGAM1 in cancer cells accelerates CtIP degradation through deprivation of the intracellular deoxyribonucleotide triphosphate pool and associated activation of the p53/p73 pathway. Enzymatic inhibition of PGAM1 decreases CtIP protein levels, impairs HR repair, and hence sensitizes BRCA1/2-proficient breast cancer to poly(ADP-ribose) polymerase (PARP) inhibitors. Together, this study identifies a metabolically dependent function of PGAM1 in promoting HR repair and reveals a potential therapeutic opportunity for PGAM1 inhibitors in combination with PARP inhibitors.
Yasuo Igarashi - One of the best experts on this subject based on the ideXlab platform.
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structural units important for activity of a novel type phosphoserine phosphatase from hydrogenobacter thermophilus tk 6 revealed by crystal structure analysis
Journal of Biological Chemistry, 2013Co-Authors: Yoko Chiba, Shoichiro Horita, Jun Ohtsuka, Masaru Tanokura, Koji Nagata, Hiroyuki Arai, Yasuo Igarashi, Masaharu IshiiAbstract:Novel-type serine-synthesizing enzymes, termed metal-independent phosphoserine phosphatases (iPSPs), were recently identified and characterized from Hydrogenobacter thermophilus, a chemolithoautotrophic bacterium belonging to the order Aquificales. iPSPs are cofactor-dependent Phosphoglycerate Mutase (dPGM)-like phosphatases that have significant amino acid sequence similarity to dPGMs but lack Phosphoglycerate Mutase activity. Genes coding dPGM-like phosphatases have been identified in a broad range of organisms; however, predicting the function of the corresponding proteins based on sequence information alone is difficult due to their diverse substrate preferences. Here, we determined the crystal structure of iPSP1 from H. thermophilus in the apo-form and in complex with its substrate l-phosphoserine to find structural units important for its phosphatase activity toward l-phosphoserine. Structural and biochemical characterization of iPSP1 revealed that the side chains of His85 and C-terminal region characteristic of iPSP1 are responsible for the PSP activity. The importance of these structural units for PSP activity was confirmed by high PSP activity observed in two novel dPGM-like proteins from Cyanobacteria and Chloroflexus in which the two structural units were conserved. We anticipate that our present findings will facilitate understanding of the serine biosynthesis pathways of organisms that lack gene(s) encoding conventional PSPs, as the structural information revealed here will help to identify iPSP from sequence databases.
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discovery and analysis of cofactor dependent Phosphoglycerate Mutase homologs as novel phosphoserine phosphatases in hydrogenobacter thermophilus
Journal of Biological Chemistry, 2012Co-Authors: Yoko Chiba, Kenro Oshima, Masaharu Ishii, Hiroyuki Arai, Yasuo IgarashiAbstract:Abstract Phosphoserine phosphatase (PSP) catalyzes the dephosphorylation of phosphoserine to serine and inorganic phosphate. PSPs, which have been found in all three domains of life, belong to the haloacid dehalogenase-like hydrolase superfamily. However, certain organisms, particularly bacteria, lack a classical PSP gene, although they appear to possess a functional phosphoserine synthetic pathway. The apparent lack of a PSP ortholog in Hydrogenobacter thermophilus, an obligately chemolithoautotrophic and thermophilic bacterium, represented a missing link in serine anabolism because our previous study suggested that serine should be synthesized from phosphoserine. Here, we detected PSP activity in cell-free extracts of H. thermophilus and purified two proteins with PSP activity. Surprisingly, these proteins belonged to the histidine phosphatase superfamily and had been annotated as cofactor-dependent Phosphoglycerate Mutase (dPGM). However, because they possessed neither Mutase activity nor the residues important for the activity, we defined these proteins as novel-type PSPs. Considering the strict substrate specificity toward l-phosphoserine, kinetic parameters, and PSP activity levels in cell-free extracts, these proteins were strongly suggested to function as PSPs in vivo. We also detected PSP activity from “dPGM-like” proteins of Thermus thermophilus and Arabidopsis thaliana, suggesting that PSP activity catalyzed by dPGM-like proteins may be distributed among a broad range of organisms. In fact, a number of bacterial genera, including Firmicutes and Cyanobacteria, were proposed to be strong candidates for possessing this novel type of PSP. These findings will help to identify the missing link in serine anabolism.