Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Cloning, sequencing, expression, purification and preliminary characterization of a type II dehydroquinase from Helicobacter pylori.

ABSTRACT: A heat-stable dehydroquinase was purified to near homogeneity from a plate-grown suspension of the Gram-negative stomach pathogen Helicobacter pylori, and shown from both its subunit and native molecular masses to be a member of the type II family of dehydroquinases. This was confirmed by N-terminal amino acid sequence data. The gene encoding this activity was isolated following initial identification, by random sequencing of the H. pylori genome, of a 96 bp fragment, the translated sequence of which showed strong identity to a C-terminal region of other type II enzymes. Southern blot analysis of a cosmid library identified several potential clones, one of which complemented an Escherichia coli aroD point mutant strain deficient in host dehydroquinase. The gene encoding the H. pylori type II dehydroquinase (designated aroQ) was sequenced. The translated sequence was identical to the N-terminal sequence obtained directly from the purified protein, and showed strong identity to other members of the type II family of dehydroquinases. The enzyme was readily expressed in E. coli from a plasmid construct from which several milligrams of protein could be isolated, and the molecular mass of the protein was confirmed by electrospray MS. The aroQ gene in H. pylori may function in the central biosynthetic shikimate pathway of this bacterium, thus opening the way for the construction of attenuated strains as potential vaccines as well as offering a new target for selective enzyme inhibition.

SUBMITTER: Bottomley JR

PROVIDER: S-EPMC1217804 | biostudies-other | 1996 Oct

REPOSITORIES: biostudies-other

ACCESS DATA

Json Xml

Similar Datasets

Cloning, purification and preliminary crystallographic analysis of the Helicobacter pylori leucyl aminopeptidase-bestatin complex.

Project description:Helicobacter pylori is an important human pathogenic bacterium associated with numerous severe gastroduodenal diseases, including ulcers and gastric cancer. Cytosolic leucyl aminopeptidase (LAP) is an important housekeeping protein that is involved in peptide and protein turnover, catabolism of proteins and modulation of gene expression. LAP is upregulated in metronidazole-resistant H. pylori, which suggests that, in addition to having an important housekeeping role, LAP contributes to the mechanism of drug resistance. Crystals of H. pylori LAP have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals belonged to the primitive triclinic space group P1, with unit-cell parameters a = 97.5, b = 100.2, c = 100.4?Å, ? = 75.4, ? = 60.9, ? = 81.8°. An X-ray diffraction data set was collected to 2.8?Å resolution from a single crystal. Molecular-replacement results using these data indicate that H. pylori LAP is a hexamer with 32 symmetry.

| S-EPMC3758151 | biostudies-literature

Cloning, purification and preliminary crystallographic analysis of the complex of Helicobacter pylori ?-carbonic anhydrase with acetazolamide.

Project description:Helicobacter pylori infection of the stomach can lead to severe gastroduodenal diseases such as gastritis, peptic ulcers and gastric cancers. Periplasmic H. pylori ?-carbonic anhydrase (Hp?CA) is essential for the acclimatization of the bacterium to the acidity of the stomach. Through the action of urease and carbonic anhydrases, the H. pylori periplasmic pH is maintained at around 6 in an environment with a pH as low as 2, which in turn facilitates the maintenance of a cytoplasmic pH close to neutral, allowing growth in the gastric niche. Crystals of Hp?CA in complex with the inhibitor acetazolamide have been grown by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitating agent. The crystals have the symmetry of space group P2(1)2(1)2(1), with unit-cell parameters a = 37.0, b = 82.4, c = 150.8?Å. An X-ray diffraction data set was collected from a single crystal to 1.7?Å resolution. Calculation of the self-rotation function using this data and molecular replacement showed that the asymmetric unit contains an Hp?CA dimer.

| S-EPMC3818046 | biostudies-literature

Cloning, purification and preliminary X-ray analysis of the C-terminal domain of Helicobacter pylori MotB.

Project description:The C-terminal domain of MotB (MotB-C) contains a putative peptidoglycan-binding motif and is believed to anchor the MotA/MotB stator unit of the bacterial flagellar motor to the cell wall. Crystals of Helicobacter pylori MotB-C (138 amino-acid residues) were obtained by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. These crystals belong to space group P2(1), with unit-cell parameters a = 50.8, b = 89.5, c = 66.3 A, beta = 112.5 degrees . The crystals diffract X-rays to at least 1.6 A resolution using a synchrotron-radiation source. Self-rotation function and Matthews coefficient calculations suggest that the asymmetric unit contains one tetramer with 222 point-group symmetry. The anomalous difference Patterson maps calculated for an ytterbium-derivative crystal using diffraction data at a wavelength of 1.38 A showed significant peaks on the v = 1/2 Harker section, suggesting that ab initio phase information could be derived from the MAD data.

| S-EPMC2374257 | biostudies-literature

Cloning, purification and preliminary crystallographic analysis of the Helicobacter pylori pseudaminic acid biosynthesis N-acetyltransferase PseH.

Project description:Helicobacter pylori infection is the common cause of gastritis and duodenal and stomach ulcers, which have been linked to a higher risk of the development of gastric cancer. The motility that facilitates persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-?-L-altropyranose. Crystals of H. pylori PseH have been grown by the hanging-drop vapour-diffusion method using diammonium tartrate as a precipitating agent. The crystals belonged to space group I222 or I212121, with unit-cell parameters a = 107.8, b = 145.4, c = 166.3?Å. A complete X-ray diffraction data set has been collected to 2.5?Å resolution using cryocooling conditions and synchrotron radiation.

| S-EPMC4157436 | biostudies-literature

Purification of Helicobacter pylori superoxide dismutase and cloning and sequencing of the gene.

Project description:The superoxide dismutase (SOD) of Helicobacter pylori, a pathogenic bacterium which colonizes the gastric mucosa, evoking a marked inflammatory response, was purified and characterized, and the N-terminal amino acid sequence was determined. The enzyme consists of two identical subunits each with an apparent molecular weight of 24,000. Analysis of the primary structure and inhibition studies revealed that H. pylori possesses a typical procaryotic iron-containing enzyme. No other isoenzymes could be detected. Indirect gold immunostaining of H. pylori SOD with a polyclonal antibody directed against the iron-containing SOD of Escherichia coli showed a surface-associated localization of the enzyme. The H. pylori SOD gene was cloned by functional complementation of a SOD-deficient E. coli mutant. Sequencing and alignment revealed striking homology to the following facultative intracellular human pathogens: Listeria ivanovii, Listeria monocytogenes, Coxiella burnetti, Porphyromonas gingivalis, Legionella pneumophila, and Entamoeba histolytica. An open reading frame of 642 bp encoding 214 amino acids was determined. There was no leader sequence detectable. Cloning of the H. pylori SOD gene is one of the prerequisites to investigation of its pathophysiological role in the defense against antimicrobial mechanisms of polymorphonuclear granulocytes.

| S-EPMC281317 | biostudies-other

Cloning, overexpression, purification and preliminary crystallographic studies of a mitochondrial type II peroxiredoxin from Pisum sativum.

Project description:A cDNA encoding an open reading frame of 199 amino acids corresponding to a type II peroxiredoxin from Pisum sativum with its transit peptide was isolated by RT-PCR. The 171-amino-acid mature protein (estimated molecular weight 18.6 kDa) was cloned into the pET3d vector and overexpressed in Escherichia coli. The recombinant protein was purified and crystallized by the hanging-drop vapour-diffusion technique. A full data set (98.2% completeness) was collected using a rotating-anode generator to a resolution of 2.8 angstroms from a single crystal flash-cooled at 100 K. X-ray data revealed that the protein crystallizes in space group P1, with unit-cell parameters a = 61.88, b = 66.40, c = 77.23 angstroms, alpha = 102.90, beta = 104.40, gamma = 99.07 degrees, and molecular replacement using a theoretical model predicted from the primary structure as a search model confirmed the presence of six molecules in the unit cell as expected from the Matthews coefficient. Refinement of the structure is in progress.

| S-EPMC2242941 | biostudies-literature

Type II dehydroquinase: molecular replacement with many copies.

Project description:Type II dehydroquinase is a small (150-amino-acid) protein which in solution packs together to form a dodecamer with 23 cubic symmetry. In crystals of this protein the symmetry of the biological unit can be coincident with the crystallographic symmetry, giving rise to cubic crystal forms with a single monomer in the asymmetric unit. In crystals where this is not the case, multiple copies of the monomer are present, giving rise to significant and often confusing noncrystallographic symmetry in low-symmetry crystal systems. These different crystal forms pose a variety of challenges for solution by molecular replacement. Three examples of structure solutions, including a highly unusual triclinic crystal form with 16 dodecamers (192 monomers) in the unit cell, are described. Four commonly used molecular-replacement packages are assessed against two of these examples, one of high symmetry and the other of low symmetry; this study highlights how program performance can vary significantly depending on the given problem. In addition, the final refined structure of the 16-dodecamer triclinic crystal form is analysed and shown not to be a superlattice structure, but rather an F-centred cubic crystal with frustrated crystallographic symmetry.

| S-EPMC2394815 | biostudies-literature

Structure of type II dehydroquinase from Pseudomonas aeruginosa.

Project description:Pseudomonas aeruginosa causes opportunistic infections and is resistant to most antibiotics. Ongoing efforts to generate much-needed new antibiotics include targeting enzymes that are vital for P. aeruginosa but are absent in mammals. One such enzyme, type II dehydroquinase (DHQase), catalyzes the interconversion of 3-dehydroquinate and 3-dehydroshikimate, a necessary step in the shikimate pathway. This step is vital for the proper synthesis of phenylalanine, tryptophan, tyrosine and other aromatic metabolites. The recombinant expression, purification and crystal structure of catalytically active DHQase from P. aeruginosa (PaDHQase) are presented. Cubic crystals belonging to space group F23, with unit-cell parameters a=b=c=125.39 Å, were obtained by vapor diffusion in sitting drops and the structure was refined to an R factor of 16% at 1.74 Å resolution. PaDHQase is a prototypical type II DHQase with the classical flavodoxin-like α/β topology.

| S-EPMC4231849 | biostudies-literature

Cloning and sequencing of cagA gene fragment of Helicobacter pylori with coccoid form.

Project description:AimTo clone and sequence the cagA gene fragment of Helicobacter pylori (H pylori) with coccoid form.MethodsH pylori strain NCTC11637 were transformed to coccoid form by exposure to antibiotics in subinhibitory concentrations. The coccoid H pylori was collected. cagA gene of the coccoid H pylori strain was amplified by PCR. After purified, the target fragment was cloned into plasmid pMD-18T. The recombinant plasmid pMD-18T-cagA was transformed into E.coli JM109. Positive clones were screened and identified by PCR and digestion with restriction endonucleases. The sequence of inserted fragment was then analysed.ResultscagA gene of 3,444 bp was obtained from the coccoid H pylori genome DNA. The recombinant plasmid pMD-18T-cagA was constructed, then it was digested by BamH I+Sac I, and the product of digestion was identical with the predicted one. Sequence analysis showed that the homology of coccoid and the reported original sequence H pylori was 99.7%.ConclusionThe recombinant plasmid containing cagA gene from coccoid H pylori has been constructed successfully. The coccoid H pylori contain completed cagA gene, which may be related to pathogenicity of them.

| S-EPMC4576237 | biostudies-literature

Expression, purification and characterization of arginase from Helicobacter pylori in its apo form.

Project description:Arginase, a manganese-dependent enzyme that widely distributed in almost all creatures, is a urea cycle enzyme that catalyzes the hydrolysis of L-arginine to generate L-ornithine and urea. Compared with the well-studied arginases from animals and yeast, only a few eubacterial arginases have been characterized, such as those from H. pylori and B. anthracis. However, these enzymes used for arginase activity assay were all expressed with LB medium, as low concentration of Mn(2+) was detectable in the medium, protein obtained were partially Mn(2+) bonded, which may affect the results of arginase activity assay. In the present study, H. pylori arginase (RocF) was expressed in a Mn(2+) and Co(2+) free minimal medium, the resulting protein was purified through affinity and gel filtration chromatography and the apo-form of RocF was confirmed by flame photometry analysis. Gel filtration indicates that the enzyme exists as monomer in solution, which was unique as compared with homologous enzymes. Arginase activity assay revealed that apo-RocF had an acidic pH optimum of 6.4 and exhibited metal preference of Co(2+)>Ni(2+)>Mn(2+). We also confirmed that heat-activation and reducing regents have significant impact on arginase activity of RocF, and inhibits S-(2-boronoethyl)-L-Cysteine (BEC) and Nω-hydroxy-nor-Arginine (nor-NOHA) inhibit the activity of RocF in a dose-dependent manner.

| S-EPMC3197605 | biostudies-literature

OmicsDI is part of the ELIXIR infrastructure

OmicsDI is an Elixir interoperability service. Learn more ›

Tweets

OmicsDI Databases

PRIDE
PeptideAtlas
MassIVE
JPOST Repository
Physiome Model Repository

EGA
EVA
ENA
LINCS
PAXDB
Cell Collective

MetaboLights
Metabolomics Workbench
MetabolomeExpress
GNPS
BioModels
FAIRDOMHub

ArrayExpress
dbGaP
ExpressionAtlas
GEO
NODE

Information

Databases
Help
API
Contact us
Code on GitHub
Terms of use
Submit Data