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Antimicrobial Agents and Chemotherapy Nov 1990Aminoglycoside-resistant strains of Staphylococcus and Enterococcus, approximately 500 of each, were screened by dot blot hybridization for the presence of genes...
Aminoglycoside-resistant strains of Staphylococcus and Enterococcus, approximately 500 of each, were screened by dot blot hybridization for the presence of genes encoding aminoglycoside-modifying enzymes. The MICs of various aminoglycosides for the strains were determined, and the enzyme contents of the cells were inferred from the resistance phenotypes. The agreements (in percent) of the hybridization results with the deduced enzyme contents for Staphylococcus and Enterococcus species were, respectively, 80 and 87.6 for ANT(6) (aminoglycoside nucleotidyltransferase), 99.8 and 100 for both APH(3') (aminoglycoside phosphotransferase) and APH(2")-AAC(6') (aminoglycoside acetyltransferase), and 100 and 100 for ANT(4'). The weak correlation obtained with the probe for ANT(6) was due to the fact that gram-positive cocci can also be streptomycin resistant by synthesis of APH(3") or ANT(3")(9) and by ribosomal mutation. The remaining probes appeared to be specific: they hybridized with all the resistant clinical isolates but not with the susceptible strains. These results indicate that, except for streptomycin, nucleic acid hybridization is a valid approach for the detection and characterization of aminoglycoside resistance in gram-positive cocci.
Topics: Acetyltransferases; Aminoglycosides; Anti-Bacterial Agents; DNA Probes; DNA, Bacterial; Drug Resistance, Microbial; France; Kanamycin Kinase; Nucleic Acid Hybridization; Nucleotidyltransferases; Phenotype; Phosphotransferases; Staphylococcus; Streptococcus
PubMed: 1963528
DOI: 10.1128/AAC.34.11.2164 -
The Journal of Biological Chemistry Apr 2002A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The...
A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively. The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.
Topics: Amino Acid Sequence; Base Sequence; Catalysis; DNA Primers; Glucokinase; Methanococcus; Molecular Sequence Data; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Phylogeny; Recombinant Proteins; Sequence Homology, Amino Acid
PubMed: 11856730
DOI: 10.1074/jbc.C200059200 -
Plant Cell Reports Jun 2016Decreased PFPase activity in rice perturbs the equilibration of carbon metabolism during grain filling but has no visible phenotypic effects during the vegetative and...
Decreased PFPase activity in rice perturbs the equilibration of carbon metabolism during grain filling but has no visible phenotypic effects during the vegetative and reproductive growth stages. Starch is a primary energy reserve for various metabolic processes in plant. Despite much advance has been achieved in pathways involved in starch biosynthesis, information was still lacked for precise regulation related to carbon metabolism during seed filling in rice (Oryza sativa). The objective of this study was to identify and characterize new gene associated with carbon metabolism during grain filling. By screening our chemical mutant pool, two allelic mutants exhibiting floury endosperm were isolated. No visible phenotypic defects were observed during both the vegetative and reproductive growth stages, except for the floury-like endosperm of grains with significantly reduced kernel thickness, 1000-grain weight and total starch content. Map-based cloning revealed that the mutant phenotypes were controlled by a gene encoding pyrophosphate: fructose-6-phosphate 1-phosphotransferase (PFP, EC 2.7.1.90) β subunit (PFPβ), which catalyzes reversible interconversion between fructose-6-phosphate and fructose-1, 6-bisphosphate. The identity of PFP β was further confirmed by a genetic complementation test. Subcellular analysis demonstrated that PFPβ was localized in cytoplasm. Quantitative PCR and histochemical staining indicated PFP β was ubiquitously expressed in various tissues. Furthermore, we found PFP β could express in both the early and late phases of starch accumulation during grain filling and decreased activity of PFP β in pfp mutants resulted in compromised carbon metabolism with increased soluble sugar contents and unfavorable starch biosynthesis. Our results highlight PFPβ functions in modulating carbon metabolism during grain filling stage.
Topics: Carbon; Cloning, Molecular; Edible Grain; Endosperm; Microscopy, Electron, Scanning; Oryza; Phosphotransferases; Phylogeny; Real-Time Polymerase Chain Reaction
PubMed: 26993329
DOI: 10.1007/s00299-016-1964-4 -
European Journal of Biochemistry Apr 1991This work was carried out to investigate the relative roles of phosphofructokinase and pyrophosphate-fructose-6-phosphate 1-phosphotransferase during the increased...
This work was carried out to investigate the relative roles of phosphofructokinase and pyrophosphate-fructose-6-phosphate 1-phosphotransferase during the increased glycolysis at the climacteric in ripening bananas (Musa cavendishii Lamb ex Paxton). Fruit were ripened in the dark in a continuous stream of air in the absence of ethylene. CO2 production, the contents of glucose 6-phosphate, fructose 6-phosphate, fructose 1,6-bisphosphate, phosphoenolpyruvate and PPi; and the maximum catalytic activities of pyrophosphate-fructose-6-phosphate 1-phosphotransferase, 6-phosphofructokinase, pyruvate kinase and phosphoenolpyruvate carboxylase were measured over a 12-day period that included the climacteric. Cytosolic fructose-1,6- bisphosphatase could not be detected in extracts of climacteric fruit. The peak of CO2 production was preceded by a threefold rise in phosphofructokinase, and accompanied by falls in fructose 6-phosphate and glucose 6-phosphate, and a rise in fructose 1,6-bisphosphate. No change in pyrophosphate-fructose-6-phosphate 1-phosphotransferase or pyrophosphate was found. It is argued that phosphofructokinase is primarily responsible for the increased entry of fructose 6-phosphate into glycolysis at the climacteric.
Topics: Carbon Dioxide; Fruit; Glycolysis; Kinetics; Phosphoenolpyruvate Carboxylase; Phosphofructokinase-1; Phosphotransferases; Plants; Pyruvate Kinase
PubMed: 1849821
DOI: 10.1111/j.1432-1033.1991.tb15907.x -
The Biochemical Journal Aug 1998Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation...
Rad, Gem and Kir possess unique structural features in comparison with other Ras-like GTPases, including a C-terminal 31-residue extension that lacks typical prenylation motifs. We have recently shown that Rad and Gem bind calmodulin in a Ca2+-dependent manner via this C-terminal extension, involving residues 278-297 in human Rad. This domain also contains several consensus sites for serine phosphorylation, and Rad is complexed with calmodulin-dependent protein kinase II (CaMKII) in C2C12 cells. Here we show that Rad serves as a substrate for phosphorylation by CaMKII, cAMP-dependent protein kinase (PKA), protein kinase C (PKC) and casein kinase II (CKII) with stoichiometries in vitro of 0.2-1.3 mol of phosphate/mol of Rad. By deletion and point mutation analysis we show that phosphorylation by CaMKII and PKA occurs on a single serine residue at position 273, whereas PKC and CKII phosphorylate multiple C-terminal serine residues, including Ser214, Ser257, Ser273, Ser290 and Ser299. Incubation of Rad with PKA decreases GTP binding by 60-70%, but this effect seems to be independent of phosphorylation, as it is observed with the Ser273-->Ala mutant of Rad containing a mutation at the site of PKA phosphorylation. The remainder of the serine kinases have no effect on Rad GTP binding, intrinsic GTP hydrolysis or GTP hydrolysis stimulated by the putative tumour metastasis suppressor nm23. However, phosphorylation of Rad by PKC and CKII abolishes the interaction of Rad with calmodulin. These findings suggest that the binding of Rad to calmodulin, as well as its ability to bind GTP, might be regulated by the activation of several serine kinases.
Topics: Binding Sites; Calcium-Calmodulin-Dependent Protein Kinase Type 2; Calcium-Calmodulin-Dependent Protein Kinases; Calmodulin; Casein Kinase II; Cyclic AMP-Dependent Protein Kinases; GTP-Binding Proteins; Guanosine Triphosphate; Humans; Hydrolysis; Kinetics; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Protein Kinase C; Protein Serine-Threonine Kinases; ras Proteins
PubMed: 9677319
DOI: 10.1042/bj3330609 -
Nature Communications Apr 2016The alarmone (p)ppGpp is commonly used by bacteria to quickly respond to nutrient starvation. Although (p)ppGpp synthetases such as SpoT have been extensively studied,...
The alarmone (p)ppGpp is commonly used by bacteria to quickly respond to nutrient starvation. Although (p)ppGpp synthetases such as SpoT have been extensively studied, little is known about the molecular mechanisms stimulating alarmone synthesis upon starvation. Here, we describe an essential role of the nitrogen-related phosphotransferase system (PTS(Ntr)) in controlling (p)ppGpp accumulation in Caulobacter crescentus. We show that cells sense nitrogen starvation by way of detecting glutamine deprivation using the first enzyme (EI(Ntr)) of PTS(Ntr). Decreasing intracellular glutamine concentration triggers phosphorylation of EI(Ntr) and its downstream components HPr and EIIA(Ntr). Once phosphorylated, both HPr∼P and EIIA(Ntr)∼P stimulate (p)ppGpp accumulation by modulating SpoT activities. This burst of second messenger primarily impacts the non-replicative phase of the cell cycle by extending the G1 phase. This work highlights a new role for bacterial PTS systems in stimulating (p)ppGpp accumulation in response to metabolic cues and in controlling cell cycle progression and cell growth.
Topics: Bacterial Proteins; Caulobacter crescentus; Cell Cycle; Gene Expression Regulation, Bacterial; Glutamine; Guanosine Pentaphosphate; Nitrogen; Phosphotransferases
PubMed: 27109061
DOI: 10.1038/ncomms11423 -
European Journal of Biochemistry Apr 2002We have investigated the crr gene of Streptomyces coelicolor that encodes a homologue of enzyme IIAGlucose of Escherichia coli, which, as a component of the...
We have investigated the crr gene of Streptomyces coelicolor that encodes a homologue of enzyme IIAGlucose of Escherichia coli, which, as a component of the phosphoenolpyruvate-dependent sugar phosphotransferase system (PTS) plays a key role in carbon regulation by triggering glucose transport, carbon catabolite repression, and inducer exclusion. As in E. coli, the crr gene of S. coelicolor is genetically associated with the ptsI gene that encodes the general phosphotransferase enzyme I. The gene product IIACrr was overproduced, purified, and polyclonal antibodies were obtained. Western blot analysis revealed that IIACrr is expressed in vivo. The functionality of IIACrr was demonstrated by phosphoenolpyruvate-dependent phosphorylation via enzyme I and the histidine-containing phosphoryl carrier protein HPr. Phosphorylation was abolished when His72, which corresponds to the catalytic histidine of E. coli IIAGlucose, was mutated. The capacity of IIACrr to operate in sugar transport was shown by complementation of the E. coli glucose-PTS. The striking functional resemblance between IIACrr and IIAGlucose was further demonstrated by its ability to confer inducer exclusion of maltose to E. coli. A specific interaction of IIACrr with the maltose permease subunit MalK from Salmonella typhimurium was uncovered by surface plasmon resonance. These data suggest that this IIAGlucose-like protein may be involved in carbon metabolism in S. coelicolor.
Topics: Amino Acid Sequence; Biological Transport; Escherichia coli; Escherichia coli Proteins; Molecular Sequence Data; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphotransferases; Sequence Analysis, DNA; Streptomyces
PubMed: 11985592
DOI: 10.1046/j.1432-1033.2002.02864.x -
The Journal of Biological Chemistry Feb 1977Galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase) was purified to homogeneity with a 50% yield from cells of Saccharomyces cerevisiae which were fully...
Galactokinase (EC 2.7.1.6; ATP:D-galactose-1-phosphotransferase) was purified to homogeneity with a 50% yield from cells of Saccharomyces cerevisiae which were fully induced for the production of the galactose metabolizing enzymes. The purification was accomplished by:(a) ammonium sulfate fractionation, (b) streptomycin sulfate precipitation. (c) DEAE-cellulose chromatography, (d) hydroxylapatite chromatography, and finally (e) Bio-Gel A-0.5 m gel filtration. The resulting preparation of galactokinase was judged to be at least 95% pure by the following criteria: (a) sodium dodecyl sulfate-polyacrylamide gel electrophoresis, (b) ultracentrifuge analysis, (c) nondissociating polyacrylamide gel electrophoresis, and (d) Bio-Gel A-0.5 m gel filtration. The purified enzyme preparation was used to determine the Km values for the two substrates, galactose and ATP, which were found to be 0.60 and 0.15 mM, respectively. Vmax was also determined and found to be 3.35 mmol/h/mg. This corresponds to a turnover rate of 3350 molecules of galactose phosphorylated/min/enzyme molecule. The effect of pH on the galactokinase-catalyzed phosphorylation of galactose was determined; the results showed the pH optimum of the reaction to be in the range of pH 8.0 to 9.0. The enzyme is highly specific for galactose since galactokinase did not appear to phosphorylate any of the other sugars tested at a rate greater than 0.5% of the rate of galactose phosphorylation. Amino acid analysis was performed on the enzyme preparation and the results were used to calculate the partial specific volume (v) of 0.736. The NH2-terminal sequence was determined for the first 3 residues. The molecular weight and subunit composition were determined by ultracentrifugation and polyacrylamide gel electrophoresis under dissociating and nondissociating conditions. The data obtained indicated that galactokinase is a monomeric protein of molecular weight 58,000.
Topics: Amino Acid Sequence; Amino Acids; Galactose; Hydrogen-Ion Concentration; Kinetics; Macromolecular Substances; Molecular Weight; Phosphotransferases; Saccharomyces cerevisiae; Species Specificity
PubMed: 14144
DOI: No ID Found -
Applied and Environmental Microbiology Sep 1998The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) utilizes high-energy phosphate present in PEP to drive the uptake of several different...
The phosphoenolpyruvate (PEP)-dependent phosphotransferase system (PTS) utilizes high-energy phosphate present in PEP to drive the uptake of several different carbohydrates in bacteria. In order to examine the role of the PTS in the physiology of Listeria monocytogenes, we identified the ptsH and ptsI genes encoding the HPr and enzyme I proteins, respectively, of the PTS. Nucleotide sequence analysis indicated that the predicted proteins are nearly 70% similar to HPr and enzyme I proteins from other organisms. Purified L. monocytogenes HPr overexpressed in Escherichia coli was also capable of complementing an HPr defect in heterologous extracts of Staphylococcus aureus ptsH mutants. Additional studies of the transcriptional organization and control indicated that the ptsH and ptsI genes are organized into a transcription unit that is under the control of a consensus-like promoter and that expression of these genes is mediated by glucose availability and pH or by by-products of glucose metabolism.
Topics: Amino Acid Sequence; Bacterial Proteins; Base Sequence; Blotting, Northern; Cloning, Molecular; Gene Expression Regulation, Bacterial; Listeria monocytogenes; Molecular Sequence Data; Phosphoenolpyruvate Sugar Phosphotransferase System; Phosphotransferases (Nitrogenous Group Acceptor); Plasmids; Polymerase Chain Reaction; Promoter Regions, Genetic; Sequence Analysis, DNA; Transcription, Genetic
PubMed: 9726852
DOI: 10.1128/AEM.64.9.3147-3152.1998 -
Journal of Bacteriology Jan 2001Acetate kinase, an enzyme widely distributed in the Bacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional...
Acetate kinase, an enzyme widely distributed in the Bacteria and Archaea domains, catalyzes the phosphorylation of acetate. We have determined the three-dimensional structure of Methanosarcina thermophila acetate kinase bound to ADP through crystallography. As we previously predicted, acetate kinase contains a core fold that is topologically identical to that of the ADP-binding domains of glycerol kinase, hexokinase, the 70-kDa heat shock cognate (Hsc70), and actin. Numerous charged active-site residues are conserved within acetate kinases, but few are conserved within the phosphotransferase superfamily. The identity of the points of insertion of polypeptide segments into the core fold of the superfamily members indicates that the insertions existed in the common ancestor of the phosphotransferases. Another remarkable shared feature is the unusual, epsilon conformation of the residue that directly precedes a conserved glycine residue (Gly-331 in acetate kinase) that binds the alpha-phosphate of ADP. Structural, biochemical, and geochemical considerations indicate that an acetate kinase may be the ancestral enzyme of the ASKHA (acetate and sugar kinases/Hsc70/actin) superfamily of phosphotransferases.
Topics: Acetate Kinase; Adenosine Diphosphate; Amino Acid Sequence; Catalytic Domain; Conserved Sequence; Crystallography; Dimerization; Evolution, Molecular; Methanosarcina; Models, Molecular; Multigene Family; Organophosphates; Phosphotransferases; Protein Structure, Secondary; Protein Structure, Tertiary
PubMed: 11133963
DOI: 10.1128/JB.183.2.680-686.2001