Browse
Submit Data
Databases
API
Help

Dataset Information

0 Views

0 Connections

0 Citations

0 Reanalyses

0 Downloads

Omics score: 0

Crystallization and preliminary X-ray analysis of a UDP-MurNAc-tripeptide D-alanyl-D-alanine-adding enzyme (PaMurF) from Pseudomonas aeruginosa.

ABSTRACT: The ATP-dependent UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase MurF catalyses the last step in the cytoplasmic phase of peptidoglycan biosynthesis, which is critical in the formation of the bacterial cell wall and in the recycling of peptidoglycan intermediates. In this study, the crystallization of MurF from the Gram-negative pathogen Pseudomonas aeruginosa in the presence of its UDP-MurNAc-tripeptide substrate is reported. The crystals belonged to space group P212121, with unit-cell parameters a = 57.81, b = 87.29, c = 92.61?Å, and data were collected to 1.92?Å resolution, allowing study of the enzyme in the substrate-liganded form for the first time.

SUBMITTER: Majce V

PROVIDER: S-EPMC3660887 | biostudies-literature | 2013 May

REPOSITORIES: biostudies-literature

ACCESS DATA

Json Xml

Publications

Crystallization and preliminary X-ray analysis of a UDP-MurNAc-tripeptide D-alanyl-D-alanine-adding enzyme (PaMurF) from Pseudomonas aeruginosa.

Majce Vita V Ruane Karen M KM Gobec Stanislav S Roper David I DI

Acta crystallographica. Section F, Structural biology and crystallization communications 20130430 Pt 5

The ATP-dependent UDP-MurNAc-tripeptide:D-Ala-D-Ala ligase MurF catalyses the last step in the cytoplasmic phase of peptidoglycan biosynthesis, which is critical in the formation of the bacterial cell wall and in the recycling of peptidoglycan intermediates. In this study, the crystallization of MurF from the Gram-negative pathogen Pseudomonas aeruginosa in the presence of its UDP-MurNAc-tripeptide substrate is reported. The crystals belonged to space group P212121, with unit-cell parameters a = ...[more]

PMID: 23695563

Similar Datasets

Purification, crystallization and preliminary X-ray crystallographic analysis of the UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (MurF) from Acinetobacter baumannii.

Project description:The emergence and global spread of multidrug-resistant Acinetobacter baumannii strains are major threats to public health. Inhibition of peptidoglycan biosynthesis is an effective strategy for the development of antibiotics. The ATP-dependent UDP-N-acetylmuramoyl-tripeptide-D-alanyl-D-alanine ligase (MurF) that is responsible for the last step of peptidoglycan biosynthesis is a validated target for the development of antibiotics. Crystals of A. baumannii MurF in complex with ATP were grown by the microbatch crystallization method at 295?K. The crystals belonged to space group P322?, with unit-cell parameters a=b=85.42, c=129.86?Å. Assuming the presence of one molecule in the asymmetric unit, the solvent content was estimated to be about 54.32%.

| S-EPMC4089545 | biostudies-literature

Crystallization and preliminary X-ray study of biosynthetic alanine racemase from Pseudomonas aeruginosa PAO1.

Project description:Biosynthetic alanine racemase (AlrPA) from Pseudomonas aeruginosa PAO1 carrying a His6 tag was expressed in Escherichia coli BL21 (DE3) cells and purified by Ni(2+)-chelating affinity and anion-exchange chromatography for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 289?K in a solution consisting of 4%(v/v) Tacsimate pH 5.0, 14%(w/v) polyethylene glycol 3350 with a protein concentration of 8?mg?ml(-1). The crystal diffracted to 2.76?Å resolution and belonged to the orthorhombic space group P212121, with unit-cell parameters a = 74.12, b = 76.97, c = 154.80?Å, ? = ? = ? = 90°.

| S-EPMC4259224 | biostudies-literature

Crystallization and preliminary X-ray study of alanine dehydrogenase from Bacillus pseudofirmus OF4.

Project description:Alanine dehydrogenase (OF4Ald) from the alkaliphilic Bacillus pseudofirmus OF4 was expressed and purified with a His6 tag in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 289?K using a solution consisting of 0.1?M Tris-HCl pH 8.0, 0.2?M LiSO4, 22%(w/v) PEG 3350. X-ray diffraction data were collected to 2.8?Å resolution. The crystal belonged to the triclinic space group P1, with unit-cell parameters a = 88.04, b = 105.59, c = 120.53?Å, ? = 88.37, ? = 78.77, ? = 82.65°.

| S-EPMC3818039 | biostudies-literature

Crystallization and preliminary X-ray crystallographic study of alanyl-tRNA synthetase from the archaeon Archaeoglobus fulgidus.

Project description:In order to analyze the alanyl-tRNA synthetase from the archaeon Archaeoglobus fulgidus, the N-terminal fragment lacking the dimerization domain and the C-terminal dimerization-domain fragment were each overexpressed in Escherichia coli, purified and crystallized. A 3.7 A resolution data set was collected for the N-terminal fragment. The crystal belongs to the tetragonal space group P4(1) or P4(3), with unit-cell parameters a = b = 101.15, c = 124.24 A. For the C-terminal fragment, a SeMet MAD data set was collected to 3.2 A resolution. The crystal belongs to the orthorhombic space group P222(1), with unit-cell parameters a = 124.15, b = 131.91, c = 138.68 A.

| S-EPMC2330182 | biostudies-literature

Crystallization and preliminary X-ray data analysis of beta-alanine synthase from Drosophila melanogaster.

Project description:Beta-alanine synthase catalyzes the last step in the reductive degradation pathway for uracil and thymine, which represents the main clearance route for the widely used anticancer drug 5-fluorouracil. Crystals of the recombinant enzyme from Drosophila melanogaster, which is closely related to the human enzyme, were obtained by the hanging-drop vapour-diffusion method. They diffracted to 3.3 A at a synchrotron-radiation source, belong to space group C2 (unit-cell parameters a = 278.9, b = 95.0, c = 199.3 A, beta = 125.8 degrees) and contain 8-10 molecules per asymmetric unit.

| S-EPMC2339735 | biostudies-literature

Crystallization and preliminary X-ray study of alkaline alanine racemase from Bacillus pseudofirmus OF4.

Project description:Alanine racemase (DadX(OF4)), a dimeric endogenous PLP-dependent alkaline enzyme from alkaliphilic Bacillus pseudofirmus OF4, was expressed in Escherichia coli and purified with a His(6) tag in a form suitable for X-ray crystallographic analysis. Crystals were grown by the hanging-drop vapour-diffusion method at 291 K using a solution containing 1.4 M sodium/potassium phosphate pH 8.2. The protein crystallized in space group P2(1)2(1)2(1), with two protein molecules in the asymmetric unit.

| S-EPMC2635857 | biostudies-literature

Expression, purification, crystallization and preliminary X-ray analysis of Pseudomonas aeruginosa AlgX.

Project description:AlgX is a periplasmic protein required for the production of the exopolysaccharide alginate in Pseudomonas sp. and Azotobacter vinelandii. AlgX has been overexpressed and purified and diffraction-quality crystals have been grown using iterative seeding and the hanging-drop vapor-diffusion method. The crystals grew as flat plates with unit-cell parameters a = 46.4, b = 120.6, c = 86.9 A, beta = 95.7 degrees . The crystals exhibited the symmetry of space group P2(1) and diffracted to a minimum d-spacing of 2.1 A. On the basis of the Matthews coefficient (V(M) = 2.25 A(3) Da(-1)), two molecules were estimated to be present in the asymmetric unit.

| S-EPMC2864699 | biostudies-literature

Structure-based site-directed mutagenesis of the UDP-MurNAc-pentapeptide-binding cavity of the FemX alanyl transferase from Weissella viridescens.

Project description:Weissella viridescens FemX (FemX(Wv)) belongs to the Fem family of nonribosomal peptidyl transferases that use aminoacyl-tRNA as the amino acid donor to synthesize the peptide cross-bridge found in the peptidoglycan of many species of pathogenic gram-positive bacteria. We have recently solved the crystal structure of FemX(Wv) in complex with the peptidoglycan precursor UDP-MurNAc-pentapeptide and report here the site-directed mutagenesis of nine residues located in the binding cavity for this substrate. Two substitutions, Lys36Met and Arg211Met, depressed FemX(Wv) transferase activity below detectable levels without affecting protein folding. Analogues of UDP-MurNAc-pentapeptide lacking the phosphate groups or the C-terminal D-alanyl residues were not substrates of the enzyme. These results indicate that Lys36 and Arg211 participate in a complex hydrogen bond network that connects the C-terminal D-Ala residues to the phosphate groups of UDP-MurNAc-pentapeptide and constrains the substrate in a conformation that is essential for transferase activity.

| S-EPMC1112068 | biostudies-literature

Purification, crystallization and preliminary X-ray diffraction data of UDP-galactopyranose mutase from Aspergillus fumigatus.

Project description:Aspergillus fumigatus UDP-galactopyranose mutase (AfUGM) is a potential drug target involved in the synthesis of the cell wall of this fungal pathogen. AfUGM was recombinantly produced in Escherichia coli, purified and crystallized by the sitting-drop method, producing orthorhombic crystals that diffracted to a resolution of 3.25 Å. The crystals contained four molecules per asymmetric unit and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 127.72, b = 134.30, c = 173.84 Å. Incorporation of selenomethionine was achieved, but the resulting crystals did not allow solution of the phase problem.

| S-EPMC3370916 | biostudies-literature

Crystallization and preliminary X-ray analysis of an alanine dehydrogenase from Bacillus megaterium WSH-002.

Project description:Alanine dehydrogenase (L-AlaDH) from Bacillus megaterium WSH-002 catalyses the NAD?-dependent interconversion of L-alanine and pyruvate. The enzyme was expressed in Escherichia coli BL21 (DE3) cells and purified with a His6 tag by Ni²?-chelating affinity chromatography for X-ray crystallographic analysis. Crystals were grown in a solution consisting of 0.1 M HEPES pH 8.0, 12%(w/v) polyethylene glycol 8000, 8%(v/v) ethylene glycol at a concentration of 15 mg ml?¹ purified protein. The crystal diffracted to 2.35 Å resolution and belonged to the trigonal space group R32, with unit-cell parameters a = b = 125.918, c = 144.698 Å.

| S-EPMC3729178 | 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