scholarly journals Expression, purification, crystallization and preliminary X-ray analysis of the PaaI-like thioesterase SAV0944 fromStaphylococcus aureus

2014 ◽  
Vol 70 (2) ◽  
pp. 244-247
Author(s):  
Yogesh B. Khandokar ◽  
Noelia Roman ◽  
Kate M. Smith ◽  
Parul Srivastava ◽  
Jade K. Forwood
Keyword(s):  
Recombinant Expression ◽  
Food Poisoning ◽  
Initial Structure ◽  
Molecular Replacement ◽  
Toxic Shock ◽  
Cellular Functions ◽  
Cell Parameters ◽  
Diffusion Technique ◽  
Peg 4000

Staphylococcus aureusis the causative agent of many diseases, including meningitis, bacteraemia, pneumonia, food poisoning and toxic shock syndrome. Structural characterization of the PaaI-like thioesterase SAV0944 (SaPaaI) fromS. aureussubsp.aureusMu50 will aid in understanding its potential as a new therapeutic target by knowledge of its molecular details and cellular functions. Here, the recombinant expression, purification and crystallization ofSaPaaI thioesterase fromS. aureusare reported. This protein initially crystallized with the ligand coenzyme A using the hanging-drop vapour-diffusion technique with condition No. 40 of Crystal Screen from Hampton Research at 296 K. Optimal final conditions consisting of 24% PEG 4000, 100 mMsodium citrate pH 6.5, 12% 2-propanol gave single diffraction-quality crystals. These crystals diffracted to beyond 2 Å resolution at the Australian Synchrotron and belonged to space groupP1211, with unit-cell parametersa= 44.05,b= 89.05,c= 60.74 Å, β = 100.5°. Initial structure determination and refinement gave anRfactor andRfreeof 17.3 and 22.0%, respectively, confirming a positive solution in obtaining phases using molecular replacement.

scholarly journals Crystallization and preliminary X-ray analysis of a novel type of lipolytic hydrolase fromBacillus licheniformis

2014 ◽  
Vol 70 (4) ◽  
pp. 473-475
Author(s):  
Hansol Ju ◽  
Ramesh Pandian ◽  
Kyungmin Kim ◽  
Kyeong Kyu Kim ◽  
T. Doohun Kim
Keyword(s):  
Structure Refinement ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Peg 4000 ◽  
Replacement Solution ◽  
Increasing Demand ◽  
Identification And Characterization

With increasing demand in biotechnological applications, the identification and characterization of novel lipolytic enzymes are of great importance. The crystallization and preliminary X-ray crystallographic study of a novel type of hydrolase fromBacillus licheniformis(BL28) are described here. Recombinant BL28 protein containing a C-terminal His tag was overproduced inEscherichia coliand purified to homogeneity. BL28 was crystallized using 0.2 Mammonium acetate, 0.1 Msodium citrate tribasic dihydrate pH 5.6, 30%(w/v) PEG 4000 as a crystallizing solution. X-ray diffraction data were collected to a resolution of 1.67 Å with anRmergeof 5.8%. The BL28 crystals belonged to the tetragonal space groupP41212, with unit-cell parametersa=b= 57.89,c= 167.25 Å. A molecular-replacement solution was obtained and structure refinement of BL28 is in progress.

Crystallization of L-aspartate oxidase, the first enzyme in the bacterial de novo biosynthesis of NAD

1999 ◽  
Vol 55 (2) ◽  
pp. 549-551 ◽  
Author(s):  
Luca Bacchella ◽  
Claudio Lina ◽  
Flavia Todone ◽  
Armando Negri ◽  
Gabriella Tedeschi ◽  
...  
Keyword(s):  
De Novo ◽  
Asymmetric Unit ◽  
Hanging Drop ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
De Novo Biosynthesis ◽  
Diffusion Technique ◽  
Peg 4000

The flavoenzyme L-aspartate oxidase from Escherichia coli was crystallized using the hanging-drop vapour-diffusion technique with PEG 4000 as precipitant. The crystals belong to space group P3121 (or P3221) with unit-cell parameters a = b = 84.9, c = 159.9 Å. A solvent content of 42% corresponds to a monomer (60 kDa) in the asymmetric unit. A complete 2.8 Å resolution data set was collected using a rotating-anode X-ray generator.

scholarly journals Characterization and structure of glyceraldehyde-3-phosphate dehydrogenase type 1 from Escherichia coli

2020 ◽  
Vol 76 (9) ◽  
pp. 406-413
Author(s):  
L. Zhang ◽  
M. R. Liu ◽  
Y. C. Yao ◽  
I. K. Bostrom ◽  
Y. D. Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Catalytic Domain ◽  
X Ray Diffraction ◽  
Cell Parameters ◽  
Diffusion Technique ◽  
Key Enzyme ◽  
Optimum Reaction ◽  
Biochemical Analyses

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a key enzyme in the glycolytic pathway that catalyzes the conversion of D-glyceraldehyde 3-phosphate to 1,3-diphosphoglycerate. Here, the full-length GAPDH type 1 from Escherichia coli (EcGAPDH1) was cloned and overexpressed, and the protein was purified. Biochemical analyses found that the optimum reaction temperature and pH of EcGAPDH1 were 55°C and 10.0, respectively. The protein has a certain amount of thermostability. Crystals of EcGAPDH1 were obtained using the sitting-drop vapor-diffusion technique and X-ray diffraction data were collected to 1.88 Å resolution. Characterization of the crystals showed that they belonged to space group P41212, with unit-cell parameters a = b = 89.651, c = 341.007 Å, α = β = γ = 90°. The structure of EcGAPDH1 contains four subunits, each of which includes an N-terminal NAD+-binding domain and a C-terminal catalytic domain. Analysis of the NAD+-bound form showed some differences between the structures of EcGAPDH1 and human GAPDH. As EcGAPDH1 shares 100% identity with GAPDH from Shigella sonnei, its structure may help in finding a drug for the treatment of shigellosis.

Structure of Azurin I from the Denitrifying Bacterium Alcaligenes xylosoxidans NCIMB 11015 at 2.45 Å Resolution

1998 ◽  
Vol 54 (3) ◽  
pp. 347-354 ◽  
Author(s):  
Chunmin Li ◽  
Tsuyoshi Inoue ◽  
Masaharu Gotowda ◽  
Shinichiro Suzuki ◽  
Kazuya Yamaguchi ◽  
...  
Keyword(s):  
Structural Model ◽  
Main Chain ◽  
Molecular Replacement ◽  
Monoclinic Crystal System ◽  
Unit Cell Parameters ◽  
Monoclinic Crystal ◽  
Cell Parameters ◽  
Replacement Method ◽  
Peg 4000 ◽  
Molecular Replacement Method

Azurin I from Alcaligenes xylosoxidans NCIMB 11015 (AzN-I) was crystallized by using PEG 4000 as a precipitant. The crystals belong to the monoclinic crystal system and have a space group C2 with the unit-cell parameters of a = 130.67, b = 54.26, c = 74.55 Å, and β = 95.99°. The structure of AzN-I has been solved by the molecular replacement method. Azurin II from the same bacterium (AzN-II) was chosen as the initial structural model. The final crystallographic R value is 17.3% and free R value is 23.6% for 10 958 reflections at a resolution of 2.45 Å. The root-mean-square deviations for main-chain atoms range between 0.19 and 0.26 Å among the four independent molecules in the asymmetric unit. The Cu atom is coordinated to Nδ of His46 and His117 at 2.0 (1) and 1.9 (1) Å, and to Sγ of Cys112 at 2.2 (1) Å, while the carbonyl O atom of Gly45 and Sδ of Met121 coordinate axially to Cu atom at 2.5 (1) and 3.1 (1) Å, respectively. The Cu—N and Cu—S distances of AzN-I are quite similar to those of AzN-II, however, the Cu—O (Gly45) bond length in AzN-I is 0.25 Å shorter than its counterpart in AzN-II. The results have been used to discuss the differences in the spectra of these two proteins.

Crystallization and preliminary X-ray diffraction studies of piratoxin II, a phospholipase A2 isolated from the venom of Bothrops pirajai

1998 ◽  
Vol 54 (6) ◽  
pp. 1437-1439
Author(s):  
W.-H. Lee ◽  
M. C. Gonçalez ◽  
R. M. F. Ramalheira ◽  
P. R. Kuser ◽  
M. H. Toyama ◽  
...  
Keyword(s):  
Crystallographic Structure ◽  
Search Model ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Diffusion Technique ◽  
Bothrops Asper ◽  
Bothrops Pirajai ◽  
Phospholipases A

The phospholipases A2 (PLA2, E.C. 3.1.1.4, phosphatide sn2 acylhydrolases) are the major components of the venom of several snakes. They are responsible for several important pharmacological effects observed in ophidian incidents. PLA2 piratoxin II from Bothrops pirajai has been crystallized by the vapour-diffusion technique. X-ray diffraction data have been collected to 2.04 Å resolution (90.2% complete, R merge = 0.070). The space group is P21 and the cell parameters are a = 46.19, b = 60.36, c = 58.74 Å and β = 96.05°. The structure has been solved by molecular replacement using the crystallographic structure of PLA2 from Bothrops asper (PDB code 1CLP) as a search model.

Purification, crystallization and preliminary crystallographic analysis of a natural complex of phospholipase A2 from Echis carinatus (saw-scaled viper)

1999 ◽  
Vol 55 (6) ◽  
pp. 1240-1241 ◽  
Author(s):  
Akanksha Nagpal ◽  
Vikas Chandra ◽  
Punit Kaur ◽  
T. P. Singh
Keyword(s):  
Crystallographic Analysis ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
Hanging Drop ◽  
Venom Protein ◽  
Cell Parameters ◽  
Molecular Weights ◽  
Unit Structure ◽  
Peg 4000 ◽  
Echis Carinatus

A novel complex of phospholipase A2 complexed with another venom protein has been isolated and purified from saw-scaled viper (Echis carinatus) venom. The molecular weights of the two components are 16 and 14 kDa, respectively. The complex was purified using an Affigel blue column and an anion-exchange (DEAE Sephacel) column. Long diamond-shaped crystals were obtained by hanging-drop vapour diffusion. The protein complex was dissolved at a concentration of 10 mg ml−1 in 20 mM sodium cacodylate, 1 mM CaCl2 and 2% dioxane at pH 6.0. The reservoir contained the same buffer with 7%(w/v) PEG 4000. Crystals appeared within 2–3 weeks. Native data to 2.9 Å resolution have been obtained at 291 K. The crystals belong to the monoclinic space group P21 with unit-cell parameters a = 74.47, b = 47.87, c = 106.39 Å, β = 104.5° and contain two molecules per asymmetric unit. Structure determination by molecular replacement is in progress.

scholarly journals Crystallization and preliminary X-ray diffraction analysis of nepenthesin-1

2014 ◽  
Vol 70 (a1) ◽  
pp. C446-C446
Author(s):  
Karla Fejfarová ◽  
Petr Man ◽  
Hynek Mrázek ◽  
Alan Kádek ◽  
Petr Halada ◽  
...  
Keyword(s):  
Ministry Of Education ◽  
X Ray Diffraction ◽  
Aspartic Proteases ◽  
Development Fund ◽  
X Ray ◽  
Cell Parameters ◽  
European Regional Development Fund ◽  
Diffusion Technique ◽  
Grant Agency

Carnivorous pitcher plants of the genus Nepenthes secrete their own aspartic proteases, nepenthesins, to digest prey. Nepenthesins differ significantly in sequence from other plant aspartic proteases. This difference, which brings more cysteine residues into the structure of nepenthesins, in conjunction with putative N-glycosylation, can contribute to uniquely high temperature and pH stabilities of these proteases [1, 2]. In continuation of our previous study of the expression and biochemical and enzymatic characterization of a recombinant form of nepenthesin-1 (rNep-1) from Nepenthes gracilis [3], we report its crystallization and preliminary X-ray analysis. Crystals of rNep-1 in complex with the pepstatin A inhibitor have been grown using the hanging-drop vapour-diffusion technique. Diffraction data were collected to 2.9 Å resolution using synchrotron radiation at Bessy II of HZB, Berlin. The crystals belong to space group P21, with unit-cell parameters a = 54.4 Å, b = 86.6 Å, c = 95.8 Å, β = 1060. The self-rotation function combined with solvent-content calculations and Matthews coefficient suggest the presence of two molecules of rNep-1 in the asymmetric unit. This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (grants No. EE2.3.30.0029 and No. LG14009), by BIOCEV CZ.1.05/1.1.00/02.0109 from the European Regional Development Fund, and by the Grant Agency of the Czech Technical University in Prague, grant No. SGS13/219/OHK4/3T/14.

Crystallization and preliminary X-ray diffraction studies of piratoxin III, a D-49 phospholipase A2 from the venom of Bothrops pirajai

1999 ◽  
Vol 55 (6) ◽  
pp. 1229-1230 ◽  
Author(s):  
Lee Wen-Hwa ◽  
Marcos H. Toyama ◽  
Andreimar M. Soares ◽  
José R. Giglio ◽  
Sérgio Marangoni ◽  
...  
Keyword(s):  
Search Model ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Diffusion Technique ◽  
Vapour Diffusion ◽  
Replacement Solution ◽  
Bothrops Pirajai

Piratoxin III (PrTX-III) is a phospholipase A2 (PLA2, E.C. 3.1.1.4, phosphatide sn-2 acylhydrolase) isolated from Bothrops pirajai. Crystals of PrTX-III were obtained using the vapour-diffusion technique and X-ray diffraction data have been collected to 2.7 Å resolution. The enzyme was crystallized in the space group C2 with unit-cell parameters a = 60.88, b = 100.75, c = 48.19 Å, β = 123.89°. A molecular-replacement solution of the structure has been found using bothropstoxin I from the venom of B. jararacussu as a search model.

scholarly journals Characterization of Staphylococcus aureus Enterotoxin L

2003 ◽  
Vol 71 (5) ◽  
pp. 2916-2919 ◽  
Author(s):  
Paul M. Orwin ◽  
J. Ross Fitzgerald ◽  
Donald Y. M. Leung ◽  
Juan A. Gutierrez ◽  
Gregory A. Bohach ◽  
...  
Keyword(s):  
Staphylococcus Aureus ◽  
Biological Activities ◽  
Bovine Mastitis ◽  
Food Poisoning ◽  
Cell Receptor ◽  
Endotoxin Shock ◽  
Toxic Shock ◽  
Variable Regions ◽  
Β Chain

ABSTRACT Staphylococcus aureus causes a wide variety of diseases. Major virulence factors of this organism include enterotoxins (SEs) that cause both food poisoning and toxic shock syndrome. Recently, a novel SE, tentatively designated SEL, was identified in a pathogenicity island from a bovine mastitis isolate. The toxin had a molecular weight of 26,000 and an isoelectric point of 8.5. Recombinant SEL shared many biological activities with SEs, including superantigenicity, pyrogenicity, enhancement of endotoxin shock, and lethality in rabbits when administered in subcutaneous miniosmotic pumps, but the protein lacked emetic activity. T cells bearing the T-cell receptor β chain variable regions 5.1, 5.2, 6.7, 16, and 22 were significantly stimulated by recombinant SEL.

scholarly journals Protein expression, characterization, crystallization and preliminary X-ray crystallographic analysis of a Fic protein fromClostridium difficile

2014 ◽  
Vol 70 (6) ◽  
pp. 827-831 ◽  
Author(s):  
Ditte Welner ◽  
Emil Dedic ◽  
Hans C. van Leeuwen ◽  
Ed Kuijper ◽  
Morten Jannik Bjerrum ◽  
...  
Keyword(s):  
Crystallographic Analysis ◽  
Gram Negative Bacteria ◽  
Post Translational Modification ◽  
Positive Bacterium ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Diffusion Technique ◽  
Expression Characterization

Fic domains in proteins are found in abundance in nature from the simplest prokaryotes to animals. Interestingly, Fic domains found in two virulence factors of Gram-negative bacteria have recently been demonstrated to catalyse the transfer of the AMP moiety from ATP to small host GTPases. This post-translational modification has attracted considerable interest and a role for adenylylation in pathology and physiology is emerging. This work was aimed at the structural characterization of a newly identified Fic protein of the Gram-positive bacteriumClostridium difficile. A constitutively active inhibitory helix mutant ofC. difficileFic was overexpressed inEscherichia coli, purified and crystallized by the vapour-diffusion technique. Preliminary X-ray crystallographic analysis shows that the crystals diffract to at least 1.68 Å resolution at a synchrotron X-ray source. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 45.6,b= 80.8,c= 144.7 Å, α = β = γ = 90°. Two molecules per asymmetric unit corresponds to a Matthews coefficient of 2.37 Å3 Da−1and a solvent content of 48%.

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