scholarly journals Crystallization and preliminary crystallographic analysis of the first condensation domain of viomycin synthetase

2013 ◽  
Vol 69 (4) ◽  
pp. 412-415 ◽  
Author(s):  
Kristjan Bloudoff ◽  
T. Martin Schmeing
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Peptide Bond Formation ◽  
Orthorhombic Space Group ◽  
Nonribosomal Peptide ◽  
Cell Parameters ◽  
Peptide Synthetases ◽  
Condensation Domain

Nonribosomal peptide synthetases (NRPSs) are large multimodular enzymes that synthesize important secondary metabolites such as antibiotics. NRPSs follow a modular synthetic logic whereby each successive amino-acid monomer is added to the peptide chain by successive multi-domain modules. The condensation domain catalyzes the central chemical event in the synthetic cycle, peptide-bond formation, and is present in every elongation module of the NRPS. Viomycin is an antituberculosis nonribosomal peptide that is synthesized by a series of four NRPS proteins and then modified by tailoring proteins. In order to study the mechanisms of peptide-bond formation in viomycin and in NRPSs in general, a structural study of the first condensation domain of the viomycin synthetase protein VioA (VioA-C1) was initiated. The gene for VioA-C1 was cloned from genomic DNA ofStreptomyces vinaceus, expressed as an octahistidine-tagged construct and purified by column chromatography. VioA-C1 was crystallized using the sitting-drop vapor-diffusion method. X-ray diffraction data were collected on a rotating-anode source to 2.9 Å resolution. The data could be indexed in the orthorhombic space groupP212121, with unit-cell parametersa= 46.165,b= 68.335,c= 146.423 Å. There is likely to be one monomer in the asymmetric unit, giving a solvent content of 49.2% and a Matthews coefficient (VM) of 2.42 Å3 Da−1. Structural determination is in progress.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of PBPD2 fromListeria monocytogenes

2014 ◽  
Vol 70 (4) ◽  
pp. 535-537 ◽  
Author(s):  
Hyung Jin Cha ◽  
Jae-Hee Jeong ◽  
Yeon-Gil Kim
Keyword(s):  
Biosynthetic Pathway ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Low Molecular Weight ◽  
Molecular Replacement ◽  
Orthorhombic Space Group ◽  
Penicillin Binding Proteins ◽  
Cell Parameters ◽  
Replacement Method ◽  
Molecular Replacement Method

Penicillin-binding proteins (PBPs), which mediate the peptidoglycan biosynthetic pathway in the bacterial cell wall, have been intensively investigated as a target for the design of antibiotics. In this study, PBPD2, a low-molecular-weight PBP encoded bylmo2812fromListeria monocytogenes, was overexpressed inEscherichia coli, purified and crystallized at 295 K using the sitting-drop vapour-diffusion method. The crystal belonged to the primitive orthorhombic space groupP212121, with unit-cell parametersa= 37.7,b= 74.7,c= 75.1 Å, and diffracted to 1.55 Å resolution. There was one molecule in the asymmetric unit. The preliminary structure was determined by the molecular-replacement method.

scholarly journals Crystallization and preliminary X-ray analysis of the hypothetical deaminase RPB_0146 fromRhodopseudomonas palustrisHaA2

2014 ◽  
Vol 70 (11) ◽  
pp. 1560-1562
Author(s):  
Guofang Zhang ◽  
Dan Yu ◽  
Guodong Yang ◽  
Hui Dong ◽  
Tongcun Zhang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Rhodopseudomonas Palustris ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Vapour Diffusion

RPB_0146, a putative deaminase fromRhodopseudomonas palustrisHaA2, was expressed inEscherichia coliBL21 (DE3) cells and purified using a His6tag by Ni2+-chelating affinity chromatography for X-ray crystallographic analysis. Diffraction-quality crystals were grown by the hanging-drop vapour-diffusion method at 289 K and diffracted to a resolution of 2.44 Å using a wavelength of 1.000 Å at the Photon Factory (KEK), Japan. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 66.26,b= 123.94,c= 155.95 Å.

scholarly journals Peptide Bond Formation in Nonribosomal Peptide Biosynthesis

1998 ◽  
Vol 273 (35) ◽  
pp. 22773-22781 ◽  
Author(s):  
Torsten Stachelhaus ◽  
Henning D. Mootz ◽  
Veit Bergendahl ◽  
Mohamed A. Marahiel
Keyword(s):  
Bond Formation ◽  
Peptide Bond ◽  
Peptide Bond Formation ◽  
Nonribosomal Peptide ◽  
Peptide Biosynthesis

scholarly journals NRPS condensation domain–substrate studies using X-ray crystallography

2014 ◽  
Vol 70 (a1) ◽  
pp. C438-C438
Author(s):  
Kristjan Bloudoff ◽  
Thomas Schmeing
Keyword(s):  
Active Site ◽  
Streptomyces Coelicolor ◽  
Covalent Bond ◽  
Thiol Group ◽  
Peptide Bond ◽  
Cysteine Residue ◽  
Local Concentration ◽  
Peptide Bond Formation ◽  
Nonribosomal Peptide ◽  
Condensation Domain

Nonribosomal peptide synthetases (NRPSs) are a family of large multimodular enzymes that synthesize structurally and functionally diverse peptides including siderophores, toxins, agriculturally-important compounds and pharmaceutically-important compounds. The condensation (C) domain is responsible for peptide bond formation, the central chemical step in nonribosomal peptide synthesis. Here we present the crystal structure of the first condensation domain of the calcium-dependent antibiotic (CDA) synthetase (CDA-C1) from Streptomyces coelicolor soaked with a small molecule compound representing the acceptor substrate. To increase the likelihood of complex formation, we designed the compound to contain a free thiol group in order to form a covalent bond between the substrate and a cysteine residue of the CDA-C1. The tethering of the substrate to the active site mimics delivery of substrate to the active site by the NRPS PCP domain, and the disulfide bond it forms with the protein will ensure a high local concentration of substrate. Initial maps, calculated from diffraction datasets collected at the home source, indicated density corresponding to the presence of substrate at the active site. This result, along with activity assay data, will help implicate residues important to enzyme catalysis and substrate specificity. In all, these studies will help characterize C domain function in NRPSs and potentiate the use of NRPSs in bioengineering experiments to produce novel or improved therapeutics.

scholarly journals Crystallization and preliminary X-ray study of biosynthetic alanine racemase fromPseudomonas aeruginosaPAO1

2014 ◽  
Vol 70 (12) ◽  
pp. 1616-1619 ◽  
Author(s):  
Honggang Zhou ◽  
Zhenzhen Li ◽  
Guofang Zhang ◽  
Shujing Xu ◽  
Zhaona Tang ◽  
...  
Keyword(s):  
Protein Concentration ◽  
Anion Exchange Chromatography ◽  
Crystallographic Analysis ◽  
Exchange Chromatography ◽  
Alanine Racemase ◽  
Diffusion Method ◽  
Hanging Drop ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters

Biosynthetic alanine racemase (AlrPA) fromPseudomonas aeruginosaPAO1 carrying a His6tag was expressed inEscherichia coliBL21 (DE3) cells and purified by Ni2+-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 groupP212121, with unit-cell parametersa= 74.12,b= 76.97,c= 154.80 Å, α = β = γ = 90°.

Purification, crystallization and preliminary X-ray crystallographic analysis of a phospholipase A2 from Daboia russelli pulchella

1999 ◽  
Vol 55 (4) ◽  
pp. 925-926 ◽  
Author(s):  
Vikas Chandra ◽  
Akanksha Nagpal ◽  
A. Srinivasan ◽  
T. P. Singh
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
The Novel ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Sequence Identity ◽  
Class 1

Phospholipases are esterolytic enzymes which hydrolyze glycerophospholipids. The pharmacological efficiency of phospholipase A2 (PLA2) enzymes is reflected by their specificity towards a tissue or organ. The Russell's viper has been classified into two classes. Class 1 contains Viper russelli russelli, Viper russelli siamensis and Viper russelli formosensis, whereas class 2 contains Daboia russelli pulchella. The sequence identity between the PLA2s from these two classes is 47%. The novel PLA2 from Daboia russelli pulchella has been crystallized using the hanging-drop vapour-diffusion method with ammonium sulfate as precipitating agent. Crystals belong to the orthorhombic space group C2221 with unit-cell parameters a = 77.01, b = 92.29, c = 76.90 Å and two molecules in the asymmetric unit. These crystals diffract to about 2.49 Å resolution using a rotating-anode source.

scholarly journals Cloning, expression, purification, crystallization and X-ray crystallographic analysis of recombinant human C1ORF123 protein

2016 ◽  
Vol 72 (3) ◽  
pp. 207-213 ◽  
Author(s):  
Siti Nurulnabila A. Rahaman ◽  
Jastina Mat Yusop ◽  
Zeti-Azura Mohamed-Hussein ◽  
Kok Lian Ho ◽  
Aik-Hong Teh ◽  
...  
Keyword(s):  
Polycystic Ovary ◽  
Hypothetical Protein ◽  
Crystallographic Analysis ◽  
Endocrine Function ◽  
Chromosome 1 ◽  
Diffusion Method ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Chloride Hexahydrate

C1ORF123 is a human hypothetical protein found in open reading frame 123 of chromosome 1. The protein belongs to the DUF866 protein family comprising eukaryote-conserved proteins with unknown function. Recent proteomic and bioinformatic analyses identified the presence of C1ORF123 in brain, frontal cortex and synapses, as well as its involvement in endocrine function and polycystic ovary syndrome (PCOS), indicating the importance of its biological role. In order to provide a better understanding of the biological function of the human C1ORF123 protein, the characterization and analysis of recombinant C1ORF123 (rC1ORF123), including overexpression and purification, verification by mass spectrometry and a Western blot using anti-C1ORF123 antibodies, crystallization and X-ray diffraction analysis of the protein crystals, are reported here. The rC1ORF123 protein was crystallized by the hanging-drop vapor-diffusion method with a reservoir solution comprised of 20% PEG 3350, 0.2 Mmagnesium chloride hexahydrate, 0.1 Msodium citrate pH 6.5. The crystals diffracted to 1.9 Å resolution and belonged to an orthorhombic space group with unit-cell parametersa= 59.32,b= 65.35,c= 95.05 Å. The calculated Matthews coefficient (VM) value of 2.27 Å3 Da−1suggests that there are two molecules per asymmetric unit, with an estimated solvent content of 45.7%.

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