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The EMBO Journal Nov 2010The phosphotransferase system (PTS) controls preferential use of sugars in bacteria. It comprises of two general proteins, enzyme I (EI) and HPr, and various...
The phosphotransferase system (PTS) controls preferential use of sugars in bacteria. It comprises of two general proteins, enzyme I (EI) and HPr, and various sugar-specific permeases. Using fluorescence microscopy, we show here that EI and HPr localize near the Escherichia coli cell poles. Polar localization of each protein occurs independently, but HPr is released from the poles in an EI- and sugar-dependent manner. Conversely, the β-glucoside-specific permease, BglF, localizes to the cell membrane. EI, HPr and BglF control the β-glucoside utilization (bgl) operon by modulating the activity of the BglG transcription factor; BglF inactivates BglG by membrane sequestration and phosphorylation, whereas EI and HPr activate it by an unknown mechanism in response to β-glucosides availability. Using biochemical, genetic and imaging methodologies, we show that EI and HPr interact with BglG and affect its subcellular localization in a phosphorylation-independent manner. Upon sugar stimulation, BglG migrates from the cell periphery to the cytoplasm through the poles. Hence, the PTS components appear to control bgl operon expression by ushering BglG between the cellular compartments. Our results reinforce the notion that signal transduction in bacteria involves dynamic localization of proteins.
Topics: Bacterial Proteins; Blotting, Western; Cell Membrane; Cytoplasm; Escherichia coli; Escherichia coli Proteins; Glucosides; Membrane Proteins; Microscopy, Fluorescence; Operon; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation; Phosphotransferases (Nitrogenous Group Acceptor); Protein Kinases; Protein Transport; RNA-Binding Proteins; Subcellular Fractions; Two-Hybrid System Techniques
PubMed: 20924357
DOI: 10.1038/emboj.2010.240 -
Acta Crystallographica. Section F,... Jun 2012Adenylate kinases (AKs) are phosphotransferase enzymes that catalyze the interconversion of adenine nucleotides, thereby playing an important role in energy metabolism....
Adenylate kinases (AKs) are phosphotransferase enzymes that catalyze the interconversion of adenine nucleotides, thereby playing an important role in energy metabolism. In Plasmodium falciparum, three AK isoforms, namely PfAK1, PfAK2 and GTP:AMP phosphotransferase (PfGAK), have been identified. While PfAK1 and PfAK2 catalyse the conversion of ATP and AMP to two molecules of ADP, PfGAK exhibits a substrate preference for GTP and AMP and does not accept ATP as a substrate. PfGAK was cloned and expressed in Escherichia coli and purified using two-step chromatography. Brown hexagonal crystals of PfGAK were obtained and a preliminary diffraction analysis was performed. X-ray diffraction data for a single PfGAK crystal were processed to 2.9 Å resolution in space group P3(1)21 or P3(2)21, with unit-cell parameters a = b = 123.49, c = 180.82 Å, α = β = 90, γ = 120°.
Topics: Crystallization; Crystallography, X-Ray; Gene Expression; Phosphotransferases (Phosphate Group Acceptor); Plasmodium falciparum
PubMed: 22684067
DOI: 10.1107/S1744309112015862 -
Biochemistry Oct 2016Aminoglycosides (AGs) are broad-spectrum antibiotics famous for their antibacterial activity against Gram-positive and Gram-negative bacteria, as well as mycobacteria....
Aminoglycosides (AGs) are broad-spectrum antibiotics famous for their antibacterial activity against Gram-positive and Gram-negative bacteria, as well as mycobacteria. In the United States, the most prescribed AGs, including amikacin (AMK), gentamicin (GEN), and tobramycin (TOB), are vital components of the treatment for resistant bacterial infections. Arbekacin (ABK), a semisynthetic AG, is widely used for the treatment of resistant Pseudomonas aeruginosa and methicillin-resistant Staphylococcus aureus in Asia. However, the rapid emergence and development of bacterial resistance are limiting the clinical application of AG antibiotics. Of all bacterial resistance mechanisms against AGs, the acquisition of AG-modifying enzymes (AMEs) by bacteria is the most common. It was previously reported that a variant of a bifunctional AME, the 6'-N-AG acetyltransferase-Ie/2″-O-AG phosphotransferase-Ia [AAC(6')-Ie/APH(2″)-Ia], containing a D80G point mutation and a truncation after amino acid 240 modified ABK and AMK at a new position, the 4‴-amine, therefore displaying a change in regiospecificity. In this study, we aimed to verify the altered regiospecificity of this bifunctional enzyme by mutation and truncation for the potential of derivatizing AGs with chemoenzymatic reactions. With the three variant enzymes in this study that contained either mutation only (D80G), truncation only (1-240), or mutation and truncation (D80G-1-240), we characterized their activity by profiling their substrate promiscuity, determined their kinetics parameters, and performed mass spectrometry to determine how and where ABK and AMK were acetylated by these enzymes. We found that the three mutant enzymes possessed distinct acetylation regiospecificity compared to that of the bifunctional AAC(6')-Ie/APH(2″)-Ia enzyme and the functional AAC(6')-Ie domain [AAC(6')/APH(2″)-1-194].
Topics: Acetylation; Acetyltransferases; Aminoglycosides; Cloning, Molecular; Kinetics; Mass Spectrometry; Mutation; Phosphotransferases; Spectrophotometry, Ultraviolet; Substrate Specificity
PubMed: 27618454
DOI: 10.1021/acs.biochem.6b00770 -
The Journal of Antibiotics Nov 1975The aminoglycoside phosphotransferase of Pseudomonas aeruginosa 21-75 was purified by affinity chromatography using dibekacin-Sephadex 4B or lividomycin A-Sepharose 4B...
The aminoglycoside phosphotransferase of Pseudomonas aeruginosa 21-75 was purified by affinity chromatography using dibekacin-Sephadex 4B or lividomycin A-Sepharose 4B followed by DEAE Sephadex A-50 chromatography. It had activities of both the known aminoglycoside 3'-phosphotransferases I and II, and transferred phosphate from ATP to the 3'-hydroxyl group of kanamycin A, ribostamycin and butirosin A and 5-hydroxyl group of lividomycin A. This enzyme was designated aminoglycoside 3'-phosphotransferase III. It showed strong substrate inhibition by kanamycin A and ribostamycin when their concentration exceeded 6 muM. Purification and characterization of this enzyme are reported.
Topics: Aminoglycosides; Butirosin Sulfate; Kanamycin; Paromomycin; Phosphotransferases; Pseudomonas aeruginosa; Ribostamycin
PubMed: 53228
DOI: 10.7164/antibiotics.28.845 -
Proteins Apr 2013The crystal structure of Ta0880, determined at 1.91 Å resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/β/α sandwich...
The crystal structure of Ta0880, determined at 1.91 Å resolution, from Thermoplasma acidophilum revealed a dimer with each monomer composed of an α/β/α sandwich domain and a smaller lid domain. The overall fold belongs to the PfkB family of carbohydrate kinases (a family member of the Ribokinase clan) which include ribokinases, 1-phosphofructokinases, 6-phosphofructo-2-kinase, inosine/guanosine kinases, fructokinases, adenosine kinases, and many more. Based on its general fold, Ta0880 had been annotated as a ribokinase-like protein. Using a coupled pyruvate kinase/lactate dehydrogenase assay, the activity of Ta0880 was assessed against a variety of ribokinase/pfkB-like family substrates; activity was not observed for ribose, fructose-1-phosphate, or fructose-6-phosphate. Based on structural similarity with nucleoside kinases (NK) from Methanocaldococcus jannaschii (MjNK, PDB 2C49, and 2C4E) and Burkholderia thailandensis (BtNK, PDB 3B1O), nucleoside kinase activity was investigated. Ta0880 (TaNK) was confirmed to have nucleoside kinase activity with an apparent KM for guanosine of 0.21 μM and catalytic efficiency of 345,000 M(-1) s(-1) . These three NKs have significantly different substrate, phosphate donor, and cation specificities and comparisons of specificity and structure identified residues likely responsible for the nucleoside substrate selectivity. Phylogenetic analysis identified three clusters within the PfkB family and indicates that TaNK is a member of a new sub-family with broad nucleoside specificities. Proteins 2013. © 2012 Wiley Periodicals, Inc.
Topics: Amino Acid Sequence; Burkholderia; Crystallography, X-Ray; Kinetics; Methanococcales; Molecular Sequence Data; Phosphotransferases; Protein Multimerization; Protein Structure, Secondary; Sequence Alignment; Substrate Specificity; Thermoplasma
PubMed: 23161756
DOI: 10.1002/prot.24212 -
FEBS Letters Dec 2016The disease-associated hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α β γ ) catalyzes the formation of mannose 6-phosphate residues on...
The disease-associated hexameric N-acetylglucosamine (GlcNAc)-1-phosphotransferase complex (α β γ ) catalyzes the formation of mannose 6-phosphate residues on lysosomal enzymes required for efficient targeting to lysosomes. Using pull-down experiments and mutant subunits, we identified a potential loop-like region in the α-subunits comprising residues 535-588 and 645-698 involved in the binding to γ-subunits. The interaction is independent of the mannose 6-phosphate receptor homology domain but requires the N-terminal unstructured part of the γ-subunit consisting of residues 26-69. These studies provide new insights into structural requirements for the assembly of the GlcNAc-1-phosphotransferase complex, and the functions of distinct domains of the α- and γ-subunits.
Topics: Acetylglucosamine; Amino Acid Sequence; Animals; HEK293 Cells; Humans; Mutation; Phenotype; Phosphotransferases; Protein Domains; Protein Subunits
PubMed: 27736005
DOI: 10.1002/1873-3468.12456 -
Archives of Biochemistry and Biophysics Mar 2011Enzyme I(Ntr) is the first protein in the nitrogen phosphotransferase pathway. Using an array of biochemical and biophysical tools, we characterized the protein,...
Enzyme I(Ntr) is the first protein in the nitrogen phosphotransferase pathway. Using an array of biochemical and biophysical tools, we characterized the protein, compared its properties to that of EI of the carbohydrate PTS and, in addition, examined the effect of substitution of all nonexchangeable protons by deuterium (perdeuteration) on the properties of EI(Ntr). Notably, we find that the catalytic function (autophosphorylation and phosphotransfer to NPr) remains unperturbed while its stability is modulated by deuteration. In particular, the deuterated form exhibits a reduction of approximately 4°C in thermal stability, enhanced oligomerization propensity, as well as increased sensitivity to proteolysis in vitro. We investigated tertiary, secondary, and local structural changes, both in the absence and presence of PEP, using near- and far-UV circular dichroism and Trp fluorescence spectroscopy. Our data demonstrate that the aromatic residues are particularly sensitive probes for detecting effects of deuteration with an enhanced quantum yield upon PEP binding and apparent decreases in tertiary contacts for Tyr and Trp side chains. Trp mutagenesis studies showed that the region around Trp522 responds to binding of both PEP and NPr. The significance of these results in the context of structural analysis of EI(Ntr) are evaluated.
Topics: Amino Acid Sequence; Arginase; Carrier Proteins; Deuterium; Enzyme Stability; Escherichia coli; Escherichia coli Proteins; Humans; Ligands; Molecular Sequence Data; Phosphate-Binding Proteins; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphorylation; Phosphotransferases (Nitrogenous Group Acceptor); Protein Conformation; Protein Multimerization; Protein Unfolding; Temperature; alpha-Synuclein
PubMed: 21185804
DOI: 10.1016/j.abb.2010.12.022 -
Journal of Bacteriology Sep 2015Salmonella enteric serovar Typhimurium, a major cause of food-borne illness, is capable of using a variety of carbon and nitrogen sources. Fructoselysine and...
A Mannose Family Phosphotransferase System Permease and Associated Enzymes Are Required for Utilization of Fructoselysine and Glucoselysine in Salmonella enterica Serovar Typhimurium.
UNLABELLED
Salmonella enteric serovar Typhimurium, a major cause of food-borne illness, is capable of using a variety of carbon and nitrogen sources. Fructoselysine and glucoselysine are Maillard reaction products formed by the reaction of glucose or fructose, respectively, with the ε-amine group of lysine. We report here that S. Typhimurium utilizes fructoselysine and glucoselysine as carbon and nitrogen sources via a mannose family phosphotransferase (PTS) encoded by gfrABCD (glucoselysine/fructoselysine PTS components EIIA, EIIB, EIIC, and EIID; locus numbers STM14_5449 to STM14_5454 in S. Typhimurium 14028s). Genes coding for two predicted deglycases within the gfr operon, gfrE and gfrF, were required for growth with glucoselysine and fructoselysine, respectively. GfrF demonstrated fructoselysine-6-phosphate deglycase activity in a coupled enzyme assay. The biochemical and genetic analyses were consistent with a pathway in which fructoselysine and glucoselysine are phosphorylated at the C-6 position of the sugar by the GfrABCD PTS as they are transported across the membrane. The resulting fructoselysine-6-phosphate and glucoselysine-6-phosphate subsequently are cleaved by GfrF and GfrE to form lysine and glucose-6-phosphate or fructose-6-phosphate. Interestingly, although S. Typhimurium can use lysine derived from fructoselysine or glucoselysine as a sole nitrogen source, it cannot use exogenous lysine as a nitrogen source to support growth. Expression of gfrABCDEF was dependent on the alternative sigma factor RpoN (σ(54)) and an RpoN-dependent LevR-like activator, which we designated GfrR.
IMPORTANCE
Salmonella physiology has been studied intensively, but there is much we do not know regarding the repertoire of nutrients these bacteria are able to use for growth. This study shows that a previously uncharacterized PTS and associated enzymes function together to transport and catabolize fructoselysine and glucoselysine. Knowledge of the range of nutrients that Salmonella utilizes is important, as it could lead to the development of new strategies for reducing the load of Salmonella in food animals, thereby mitigating its entry into the human food supply.
Topics: Animals; Caproates; Gene Expression Regulation, Bacterial; Glucosamine; Humans; Lysine; Membrane Transport Proteins; Molecular Structure; Phosphotransferases; RNA Polymerase Sigma 54; Salmonella typhimurium; Substrate Specificity
PubMed: 26100043
DOI: 10.1128/JB.00339-15 -
PLoS Genetics Apr 2018Two-component systems constitute phosphotransfer signaling pathways and enable adaptation to environmental changes, an essential feature for bacterial survival. The...
Two-component systems constitute phosphotransfer signaling pathways and enable adaptation to environmental changes, an essential feature for bacterial survival. The general stress response (GSR) in the plant-protecting alphaproteobacterium Sphingomonas melonis Fr1 involves a two-component system consisting of multiple stress-sensing histidine kinases (Paks) and the response regulator PhyR; PhyR in turn regulates the alternative sigma factor EcfG, which controls expression of the GSR regulon. While Paks had been shown to phosphorylate PhyR in vitro, it remained unclear if and under which conditions direct phosphorylation happens in the cell, as Paks also phosphorylate the single domain response regulator SdrG, an essential yet enigmatic component of the GSR signaling pathway. Here, we analyze the role of SdrG and investigate an alternative function of the membrane-bound PhyP (here re-designated PhyT), previously assumed to act as a PhyR phosphatase. In vitro assays show that PhyT transfers a phosphoryl group from SdrG to PhyR via phosphoryl transfer on a conserved His residue. This finding, as well as complementary GSR reporter assays, indicate the participation of SdrG and PhyT in a Pak-SdrG-PhyT-PhyR phosphorelay. Furthermore, we demonstrate complex formation between PhyT and PhyR. This finding is substantiated by PhyT-dependent membrane association of PhyR in unstressed cells, while the response regulator is released from the membrane upon stress induction. Our data support a model in which PhyT sequesters PhyR, thereby favoring Pak-dependent phosphorylation of SdrG. In addition, PhyT assumes the role of the SdrG-phosphotransferase to activate PhyR. Our results place SdrG into the GSR signaling cascade and uncover a dual role of PhyT in the GSR.
Topics: Phosphorylation; Phosphotransferases; Signal Transduction; Sphingomonas; Stress, Physiological
PubMed: 29652885
DOI: 10.1371/journal.pgen.1007294 -
The New Phytologist Jul 2014Two-component signalling (TCS) systems play important roles in cytokinin and ethylene signalling in Arabidopsis thaliana. Although the involvement of histidine kinases...
Two-component signalling (TCS) systems play important roles in cytokinin and ethylene signalling in Arabidopsis thaliana. Although the involvement of histidine kinases (AHKs) in drought stress responses has been described, their role and that of histidine phosphotransferases (AHPs) in guard cell signalling remain to be fully elucidated. Here, we investigated the roles of TCS genes, the histidine phosphotransferase AHP2 and the histidine kinases AHK2 and AHK3, previously reported to play roles in cytokinin and abscisic acid (ABA) signalling. We show that AHP2 is present in the nucleus and the cytoplasm, and is involved in light-induced opening. We also present evidence that there is some redistribution of AHP2 from the nucleus to the cytoplasm on addition of ABA. In addition, we provide data to support a role for the cytokinin receptors AHK2 and AHK3 in light-induced stomatal opening and, by inference, in controlling the stomatal sensitivity to ABA. Our results provide new insights into the operation of TCS in plants, cross-talk in stomatal signalling and, in particular, the process of light-induced stomatal opening.
Topics: Abscisic Acid; Arabidopsis; Arabidopsis Proteins; Cell Nucleus; Cytoplasm; Gene Expression Regulation, Plant; Histidine Kinase; Light; Phosphotransferases; Plant Cells; Plant Leaves; Plant Stomata; Plants, Genetically Modified; Protein Kinases; Signal Transduction
PubMed: 24758561
DOI: 10.1111/nph.12813