scholarly journals Pseudomonas aeruginosa Exopolyphosphatase Is Also a Polyphosphate: ADP Phosphotransferase

2015 ◽  
Vol 2015 ◽  
pp. 1-13 ◽  
Author(s):  
Paola R. Beassoni ◽  
Lucas A. Gallarato ◽  
Cristhian Boetsch ◽  
Mónica N. Garrido ◽  
Angela T. Lisa
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Enzyme Activity ◽  
Active Site ◽  
Inorganic Phosphate ◽  
Biochemical Characterization ◽  
E Coli ◽  
Activity Dependent ◽  
Hydrolysis Of ◽  
Molecular Modeling And Docking

Pseudomonas aeruginosa exopolyphosphatase (paPpx; EC 3.6.1.11) catalyzes the hydrolysis of polyphosphates (polyP), producing polyPn−1 plus inorganic phosphate (Pi). In a recent work we have shown that paPpx is involved in the pathogenesis of P. aeruginosa. The present study was aimed at performing the biochemical characterization of this enzyme. We found some properties that were already described for E. coli Ppx (ecPpx) but we also discovered new and original characteristics of paPpx: (i) the peptide that connects subdomains II and III is essential for enzyme activity; (ii) NH4+ is an activator of the enzyme and may function at concentrations lower than those of K+; (iii) Zn2+ is also an activator of paPpx and may substitute Mg2+ in the catalytic site; and (iv) paPpx also has phosphotransferase activity, dependent on Mg2+ and capable of producing ATP regardless of the presence or absence of K+ or NH4+ ions. In addition, we detected that the active site responsible for the phosphatase activity is also responsible for the phosphotransferase activity. Through the combination of molecular modeling and docking techniques, we propose a model of the paPpx N-terminal domain in complex with a polyP chain of 7 residues long and a molecule of ADP to explain the phosphotransferase activity.

scholarly journals Biochemical Characterization of the Naturally Occurring Oxacillinase OXA-50 of Pseudomonas aeruginosa

2004 ◽  
Vol 48 (6) ◽  
pp. 2043-2048 ◽  
Author(s):  
Delphine Girlich ◽  
Thierry Naas ◽  
Patrice Nordmann
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Nucleotide Sequence ◽  
Biochemical Characterization ◽  
The Other ◽  
Multicopy Plasmid ◽  
E Coli ◽  
Naturally Occurring ◽  
Lactamase Gene

ABSTRACT The bla OXA-50 gene (formerly known as the PA5514 gene) is an oxacillinase gene identified in silico in the genome of Pseudomonas aeruginosa PAO1. By using a mutant strain of P. aeruginosa PAO1 that had an inactivated bla AmpC cephalosporinase gene, the bla OXA-50 gene was shown to be expressed constitutively in P. aeruginosa. This β-lactamase gene was cloned onto a multicopy plasmid and expressed in P. aeruginosa and Escherichia coli. It conferred decreased susceptibility to ampicillin and ticarcillin and, interestingly, to moxalactam and meropenem in P. aeruginosa but not in E. coli. Overexpression and purification enabled us to determine the molecular mass (25 kDa), the pI value (8.6), and the hydrolysis spectrum of the OXA-50 β-lactamase. It is a narrow-spectrum oxacillinase that uncommonly hydrolyzes imipenem, although at a low level. Very similar oxacillinase genes were identified in all P. aeruginosa isolates from various geographical origins tested. The weak variability of the nucleotide sequence of this gene (0 to 2%) corresponded to that found for the naturally occurring bla AmpC cephalosporinase gene of P. aeruginosa. The study indicated that P. aeruginosa harbors two naturally encoded β-lactamase genes, one of which encodes an inducible cephalosporinase and the other of which encodes a constitutively expressed oxacillinase.

scholarly journals An HD domain phosphohydrolase active site tailored for oxetanocin-A biosynthesis

2016 ◽  
Vol 113 (48) ◽  
pp. 13750-13755 ◽  
Author(s):  
Jennifer Bridwell-Rabb ◽  
Gyunghoon Kang ◽  
Aoshu Zhong ◽  
Hung-wen Liu ◽  
Catherine L. Drennan
Keyword(s):  
Signal Transduction ◽  
Crystal Structures ◽  
Bacillus Megaterium ◽  
Active Site ◽  
Inorganic Phosphate ◽  
Biochemical Characterization ◽  
Membered Ring ◽  
Nucleotide Metabolism ◽  
X Ray

HD domain phosphohydrolase enzymes are characterized by a conserved set of histidine and aspartate residues that coordinate an active site metallocenter. Despite the important roles these enzymes play in nucleotide metabolism and signal transduction, few have been both biochemically and structurally characterized. Here, we present X-ray crystal structures and biochemical characterization of theBacillus megateriumHD domain phosphohydrolase OxsA, involved in the biosynthesis of the antitumor, antiviral, and antibacterial compound oxetanocin-A. These studies reveal a previously uncharacterized reaction for this family; OxsA catalyzes the conversion of a triphosphorylated compound into a nucleoside, releasing one molecule of inorganic phosphate at a time. Remarkably, this functionality is a result of the OxsA active site, which based on structural and kinetic analyses has been tailored to bind the small, four-membered ring of oxetanocin-A over larger substrates. Furthermore, our OxsA structures show an active site that switches from a dinuclear to a mononuclear metal center as phosphates are eliminated from substrate.

scholarly journals Biophysical characterization of two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2

2020 ◽  
Author(s):  
Talita Stelling de Araújo ◽  
Sandra M. N. Scapin ◽  
William de Andrade ◽  
Maira Fasciotti ◽  
Mariana T. Q. de Magalhães ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Active Site ◽  
Lymphoblastic Leukemia ◽  
Biophysical Characterization ◽  
E Coli ◽  
Terminal Loop ◽  
Hydrolysis Of

AbstractThe hydrolysis of asparagine and glutamine by L-asparaginase has been used to treat acute lymphoblastic leukemia for over four decades. Each L-asparaginase monomer has a long loop that closes over the active site upon substrate binding, acting as a lid. Here we present a comparative study two commercially available preparations of the drug containing Escherichia coli L-Asparaginase 2, performed by a comprehensive array of biophysical and biochemical approaches. We report the oligomeric landscape and conformational and dynamic plasticity of E. coli type 2 L-asparaginase (EcA2) present in two different formulations, and its relationship with L-aspartic acid, which is present in Aginasa, but not in Leuginase. EcA2 shows a composition of monomers and oligomers up to tetramers, which is mostly not altered in the presence of L-Asp. The N-terminal loop of Leuginase, which is part of the active site is flexibly disordered, but gets ordered as in Aginasa in the presence os L-Asp, while L-Glu only does so to a limited extent. Ion-mobility spectrometry–mass spectrometry reveals two conformers for the monomeric EcA2, one of which can selectively bind to L-Asp and L-Glu. Aginasa has higher resistance to in vitro proteolysis than Leuginase, and this is directly related to the presence of L-Asp.

scholarly journals Identification and biochemical characterization of a novel PP2C-like Ser/Thr phosphatase inE. coli

2018 ◽  
Author(s):  
Krithika Rajagopalan ◽  
Jonathan Dworkin
Keyword(s):  
Biochemical Characterization ◽  
Regulatory Protein ◽  
Biochemical Properties ◽  
Bacterial Genomes ◽  
Phosphatase Inhibitors ◽  
E Coli ◽  
Specificity And Sensitivity ◽  
Genes Encoding ◽  
Number Of Bacteria

AbstractIn bacteria, signaling phosphorylation is thought to occur primarily on His and Asp residues. However, phosphoproteomic surveys in phylogenetically diverse bacteria over the past decade have identified numerous proteins that are phosphorylated on Ser and/or Thr residues. Consistently, genes encoding Ser/Thr kinases are present in many bacterial genomes such asE. coli,which encodes at least three Ser/Thr kinases. Since Ser/Thr phosphorylation is a stable modification, a dedicated phosphatase is necessary to allow reversible regulation. Ser/Thr phosphatases belonging to several conserved families are found in bacteria. One family of particular interest are Ser/Thr phosphatases which have extensive sequence and structural homology to eukaryotic Ser/Thr PP2C phosphatases. These proteins, called eSTPs (eukaryotic-like Ser/Thr phosphatases), have been identified in a number of bacteria, but not inE. coli.Here, we describe a previously unknown eSTP encoded by anE. coliORF,yegK,and characterize its biochemical properties including its kinetics, substrate specificity and sensitivity to known phosphatase inhibitors. We investigate differences in the activity of this protein in closely relatedE. colistrains. Finally, we demonstrate that this eSTP acts to dephosphorylate a novel Ser/Thr kinase which is encoded in the same operon.ImportanceRegulatory protein phosphorylation is a conserved mechanism of signaling in all biological systems. Recent phosphoproteomic analyses of phylogenetically diverse bacteria including the model Gram-negative bacteriumE. colidemonstrate that many proteins are phosphorylated on serine or threonine residues. In contrast to phosphorylation on histidine or aspartate residues, phosphorylation of serine and threonine residues is stable and requires the action of a partner Ser/Thr phosphatase to remove the modification. Although a number of Ser/Thr kinases have been reported inE. coli, no partner Ser/Thrphosphatases have been identified. Here, we biochemically characterize a novel Ser/Thr phosphatase that acts to dephosphorylate a Ser/Thr kinase that is encoded in the same operon.

scholarly journals Structural and biochemical characterization of the environmental MBLs MYO-1, ECV-1 and SHD-1

2020 ◽  
Vol 75 (9) ◽  
pp. 2554-2563 ◽  
Author(s):  
Christopher Fröhlich ◽  
Vidar Sørum ◽  
Sandra Huber ◽  
Ørjan Samuelsen ◽  
Fanny Berglund ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Homology Modelling ◽  
Catalytic Efficiency ◽  
Hydrolytic Activity ◽  
Human Pathogens ◽  
E Coli ◽  
X Ray Crystallography ◽  
Environmental Reservoir

Abstract Background MBLs form a large and heterogeneous group of bacterial enzymes conferring resistance to β-lactam antibiotics, including carbapenems. A large environmental reservoir of MBLs has been identified, which can act as a source for transfer into human pathogens. Therefore, structural investigation of environmental and clinically rare MBLs can give new insights into structure–activity relationships to explore the role of catalytic and second shell residues, which are under selective pressure. Objectives To investigate the structure and activity of the environmental subclass B1 MBLs MYO-1, SHD-1 and ECV-1. Methods The respective genes of these MBLs were cloned into vectors and expressed in Escherichia coli. Purified enzymes were characterized with respect to their catalytic efficiency (kcat/Km). The enzymatic activities and MICs were determined for a panel of different β-lactams, including penicillins, cephalosporins and carbapenems. Thermostability was measured and structures were solved using X-ray crystallography (MYO-1 and ECV-1) or generated by homology modelling (SHD-1). Results Expression of the environmental MBLs in E. coli resulted in the characteristic MBL profile, not affecting aztreonam susceptibility and decreasing susceptibility to carbapenems, cephalosporins and penicillins. The purified enzymes showed variable catalytic activity in the order of <5% to ∼70% compared with the clinically widespread NDM-1. The thermostability of ECV-1 and SHD-1 was up to 8°C higher than that of MYO-1 and NDM-1. Using solved structures and molecular modelling, we identified differences in their second shell composition, possibly responsible for their relatively low hydrolytic activity. Conclusions These results show the importance of environmental species acting as reservoirs for MBL-encoding genes.

Biochemical characterization of the tartrate-resistant acid phosphatase of human spleen with leukemic reticuloendotheliosis as a pyrophosphatase.

1977 ◽  
Vol 23 (1) ◽  
pp. 89-94 ◽  
Author(s):  
K W Lam ◽  
L T Yam
Keyword(s):  
Acid Phosphatase ◽  
Biochemical Characterization ◽  
Normal Animal ◽  
Tartrate Resistant Acid Phosphatase ◽  
Animal Tissues ◽  
Human Spleen ◽  
Optimal Activity ◽  
Catalytic Function ◽  
Hydrolysis Of

Abstract A tartrate-resistant acid phosphatase was isolated from a human leukemic spleen by freeze-thawing in saline and purified by repeated chromatography on carboxymethyl-cellulose. The purified enzyme has a molecular weight of 64 000. It catalyzes the hydrolysis of inorganic and organic pyrophosphate as well as the phenolic ester of monoorthophosphate, with optimal activity between pH 5 and 6. However, there is no activity toward mono-orthophosphate esters of aliphatic alcohols. The present data have identified its catalytic function as a pyrophosphatase. However, it has properties different from the pyrophosphatase previously observed in normal animal tissues.

scholarly journals Dissection of the Hydrogen Metabolism of the EnterobacteriumTrabulsiella guamensis: Identification of a Formate-Dependent and Essential Formate Hydrogenlyase Complex Exhibiting Phylogenetic Similarity to Complex I

2019 ◽  
Vol 201 (12) ◽  
Author(s):  
Ute Lindenstrauß ◽  
Constanze Pinske
Keyword(s):  
Fossil Fuels ◽  
Biochemical Characterization ◽  
Model Organism ◽  
Attractive Alternative ◽  
Hydrogen Metabolism ◽  
Content Type ◽  
E Coli ◽  
Formate Hydrogenlyase ◽  
Alternative Carbon Source

ABSTRACTTrabulsiella guamensisis a nonpathogenic enterobacterium that was isolated from a vacuum cleaner on the island of Guam. It has one H2-oxidizing Hyd-2-type hydrogenase (Hyd) and encodes an H2-evolving Hyd that is most similar to the uncharacterizedEscherichia coliformate hydrogenlyase (FHL-2Ec) complex. TheT. guamensisFHL-2 (FHL-2Tg) complex is predicted to have 5 membrane-integral and between 4 and 5 cytoplasmic subunits. We showed that the FHL-2Tgcomplex catalyzes the disproportionation of formate to CO2and H2. FHL-2Tghas activity similar to that of theE. coliFHL-1Eccomplex in H2evolution from formate, but the complex appears to be more labile upon cell lysis. Cloning of the entire 13-kbp FHL-2Tgoperon in the heterologousE. colihost has now enabled us to unambiguously prove FHL-2Tgactivity, and it allowed us to characterize the FHL-2Tgcomplex biochemically. Although the formate dehydrogenase (FdhH) genefdhFis not contained in the operon, the FdhH is part of the complex, and FHL-2Tgactivity was dependent on the presence ofE. coliFdhH. Also, in contrast toE. coli,T. guamensiscan ferment the alternative carbon source cellobiose, and we further investigated the participation of both the H2-oxidizing Hyd-2Tgand the H2-forming FHL-2Tgunder these conditions.IMPORTANCEBiological H2production presents an attractive alternative for fossil fuels. However, in order to compete with conventional H2production methods, the process requires our understanding on a molecular level. FHL complexes are efficient H2producers, and the prototype FHL-1Eccomplex inE. coliis well studied. This paper presents the first biochemical characterization of an FHL-2-type complex. The data presented here will enable us to solve the long-standing mystery of the FHL-2Eccomplex, allow a first biochemical characterization ofT. guamensis’s fermentative metabolism, and establish this enterobacterium as a model organism for FHL-dependent energy conservation.

Sign in / Sign up

Export Citation Format

Share Document