scholarly journals Crystallization and preliminary X-ray analysis of a highly stable novel SGNH hydrolase (Est24) fromSinorhizobium meliloti

2014 ◽  
Vol 70 (2) ◽  
pp. 193-195 ◽  
Author(s):  
Bum Han Ryu ◽  
Duy Duc Nguyen ◽  
Tri Duc Ngo ◽  
Changsuk Oh ◽  
Ramesh Pandian ◽  
...  
Keyword(s):  
Sinorhizobium Meliloti ◽  
Structure Refinement ◽  
Ammonium Phosphate ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Hydrolysis Of ◽  
Hydrolase Family

The SGNH hydrolase family includes enzymes that catalyze the hydrolysis of a broad range of substrates. Here, the crystallization and preliminary X-ray crystallographic studies of a novel SGNH hydrolase (Est24) fromSinorhizobium melilotiwere performed. Recombinant Est24 protein containing an N-terminal His tag was expressed inEscherichia coliand purified to homogeneity. Est24 was then crystallized using a solution consisting of 0.2 Mammonium phosphate pH 4.6, 20% polyethylene glycol 3350. X-ray diffraction data were collected to a resolution of 1.45 Å with anRmergeof 9.4%. The Est24 crystals belonged to space groupC2, with unit-cell parametersa= 129.09,b = 88.63,c= 86.15 Å, α = 90.00, β = 114.30, γ = 90.00°. A molecular-replacement solution was obtained using the crystal structure ofMycobacterium smegmatisarylesterase as a template and structure refinement of Est24 is in progress.

scholarly journals Expression, purification, crystallization and preliminary X-ray diffraction analysis ofAspergillus terreusendo-β-1,4-glucanase from glycoside hydrolase family 12

2014 ◽  
Vol 70 (2) ◽  
pp. 267-270 ◽  
Author(s):  
Fernando Segato ◽  
Gabriela L. Berto ◽  
Evandro Ares de Araújo ◽  
João Renato Muniz ◽  
Igor Polikarpov
Keyword(s):  
Glycoside Hydrolase ◽  
Aspergillus Terreus ◽  
Glycoside Hydrolases ◽  
Glycoside Hydrolase Family ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Hydrolase Family

Endoglucanases are important enzymes that are involved in the modification and degradation of cellulose. Filamentous fungi such asAspergillus terreusare effective biomass degraders in nature owing to their capacity to produce an enzymatic arsenal of glycoside hydrolases, including endoglucanase from glycoside hydrolase family 12 (GH12). TheA. terreusGH12 endoglucanase was cloned and overexpressed inA. nidulans, purified and crystallized. A single crystal was obtained from a solution consisting of 2 Mammonium sulfate, 5%(v/v) 2-propanol. X-ray diffraction data were collected to a resolution of 1.85 Å using synchrotron radiation and a preliminary molecular-replacement solution was obtained in the trigonal space groupP3221. The unit-cell parameters werea=b= 103.24,c= 48.96 Å.

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.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of α-glucosidase HaG fromHalomonassp. strain H11

2014 ◽  
Vol 70 (4) ◽  
pp. 464-466 ◽  
Author(s):  
Xing Shen ◽  
Wataru Saburi ◽  
Zuo-Qi Gai ◽  
Keisuke Komoda ◽  
Jian Yu ◽  
...  
Keyword(s):  
Halophilic Bacterium ◽  
Crystallographic Analysis ◽  
Low Molecular Weight ◽  
Glycoside Hydrolase Family ◽  
Molecular Replacement ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Hydrolysis Of ◽  
Hydrolase Family

The α-glucosidase HaG from the halophilic bacteriumHalomonassp. strain H11 catalyzes the hydrolysis of the glucosidic linkage at the nonreducing end of α-glucosides, such as maltose and sucrose, to release α-glucose. Based on its amino-acid sequence, this enzyme is classified as a member of glycoside hydrolase family 13. HaG has three unique characteristics: (i) a very narrow substrate specificity, almost exclusively hydrolyzing disaccharides; (ii) activation by monovalent cations, such as K+, Rb+, Cs+and NH4+; and (iii) high transfer activity of the glucose moiety to the OH group of low-molecular-weight compounds, including glycerol and 6-gingerol. Crystallographic studies have been performed in order to understand these special features. An expression vector was constructed and recombinant HaG protein was overexpressed, purified and crystallized. A data set to 2.15 Å resolution was collected and processed. The crystal belonged to space groupP212121, with unit-cell parametersa= 60.2,b= 119.2,c= 177.2 Å. The structure has been determined by molecular replacement using the isomaltulose synthase PalI as the search model (PDB entry 1m53).

scholarly journals Crystallization and preliminary X-ray diffraction studies of frutalin, an α-D-galactose-specific lectin from Artocarpus incisa seeds

2015 ◽  
Vol 71 (10) ◽  
pp. 1282-1285 ◽  
Author(s):  
Ana Cristina de Oliveira Monteiro-Moreira ◽  
Humberto D'Muniz Pereira ◽  
Antonio Eufrasio Vieira Neto ◽  
Frederico Bruno Mendes Batista Moreno ◽  
Marina Duarte Pinto Lobo ◽  
...  
Keyword(s):  
Biomarker Discovery ◽  
Spectrometric Analysis ◽  
Carbohydrate Binding ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Maximum Resolution ◽  
Replacement Solution ◽  
Artocarpus Incisa

Frutalin is an α-D-galactose-specific carbohydrate-binding glycoprotein with antitumour properties and is a powerful tool for tumour biomarker discovery. The crystallization and preliminary X-ray diffraction analysis of this lectin, which was isolated from Artocarpus incisa seeds, are reported here. Frutalin was purified and submitted to mass-spectrometric analysis. Diverse masses at approximately 16 kDa were observed in the deconvoluted spectra, which support the presence of isoforms. The best frutalin crystals were grown within a week in 0.1 M citric acid pH 3.5 which contained 25% PEG 3350 as a precipitant at 293 K, and diffracted to a maximum resolution of 1.81 Å. The monoclinic crystals belonged to space group I2, with unit-cell parameters a = 76.17, b = 74.56, c = 118.98 Å, β = 96.56°. A molecular-replacement solution was obtained which indicated the presence of four monomers per asymmetric unit. Crystallographic refinement of the structure is in progress.

Crystallization and Preliminary X-ray Diffraction Analysis of Cold-Active β-Galactosidase from Arthrobacter sp. C2-2

2005 ◽  
Vol 70 (1) ◽  
pp. 124-132 ◽  
Author(s):  
Hana Petroková ◽  
Eva Vondráčková ◽  
Tereza Skálová ◽  
Jan Dohnálek ◽  
Petra Lipovová ◽  
...  
Keyword(s):  
Structure Refinement ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
Phase Problem ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Psychrotrophic Bacteria ◽  
Cold Active

β-Galactosidase from psychrotrophic bacteria strainArthrobactersp. C2-2 catalyzes cleavage of β-D-galactosyl moieties from β-D-galactosides and is interesting for its activity at low temperatures. Various types of crystals with dimensions of up to 0.8 mm were obtained and X-ray diffraction data up to 1.9 Å were collected. The crystals belong to the monoclinic space groupP21with unit-cell parametersa= 140.1 Å,b= 205.7 Å,c= 140.5 Å and β = 102.3°. The enzyme (molecular weight of a monomer is 111 kDa) forms hexamers in the crystal structure (one hexamer per asymmetric unit). The phase problem was solved by molecular replacement. Structure refinement is in progress.

scholarly journals Purification and crystallographic analysis of a FAD-dependent halogenase fromStreptomycessp. JCM9888

2015 ◽  
Vol 71 (8) ◽  
pp. 972-976 ◽  
Author(s):  
Yanqun Zhao ◽  
Baohua Yan ◽  
Ting Yang ◽  
Jian Jiang ◽  
Heng Wei ◽  
...  
Keyword(s):  
Model Building ◽  
Structure Refinement ◽  
Crystallographic Analysis ◽  
Monoclinic Space Group ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Hexahistidine Tag ◽  
Crystallization Experiments

A new FAD (flavin adenine dinucleotide)-dependent halogenase HalY fromStreptomycessp. JCM9888 was reported to be involved in the regioselective halogenation of adenine. HalY is a variant B FAD-dependent halogenase that is most similar to the halogenase PltA involved in pyoluteorin biosynthesis. This study reports the overexpression and purification of HalY with an N-terminal hexahistidine tag, followed by crystallization experiments and X-ray crystallographic analysis. HalY was purified as a monomer in solution and crystallized to give X-ray diffraction to a resolution of 1.7 Å. The crystal belonged to the monoclinic space groupP21, with unit-cell parametersa= 41.4,b= 113.4,c= 47.6 Å, α = γ = 90, β = 107.4°, and contained one monomer of HalY in the asymmetric unit, with a calculated Matthews coefficient of 2.3 Å3 Da−1and a solvent content of 46%. The structure of the halogenase CndH was used as a search model in molecular replacement to obtain the initial model of HalY. Manual model building and structure refinement of HalY are in progress.

scholarly journals Expression, purification, crystallization and preliminary X-ray diffraction analysis of a ribokinase from the thermohalophileHalothermothrix orenii

2012 ◽  
Vol 68 (2) ◽  
pp. 240-243 ◽  
Author(s):  
Lokesh D. Kori ◽  
Andreas Hofmann ◽  
Bharat K. C. Patel
Keyword(s):  
Metal Ion ◽  
Molecular Replacement ◽  
Asymmetric Unit ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Replacement Solution ◽  
Metal Ion Affinity Chromatography

A ribokinase gene (rbk) from the anaerobic halothermophilic bacteriumHalothermothrix oreniiwas cloned and overexpressed inEscherichia coli. The recombinant protein (Ho-Rbk) was purified using immobilized metal-ion affinity chromatography and crystals were obtained using the sitting-drop method. Diffraction data were collected to a resolution of 3.1 Å using synchrotron radiation. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 45.6,b= 61.1,c= 220.2, and contained two molecules per asymmetric unit. A molecular-replacement solution has been found and attempts are currently under way to build a model of the ribokinase. Efforts to improve crystal quality so that higher resolution data can be obtained are also being considered.

scholarly journals Purification, crystallization and preliminary X-ray diffraction analysis of ArsH fromSynechocystissp. strain PCC 6803

2014 ◽  
Vol 70 (4) ◽  
pp. 497-500 ◽  
Author(s):  
Xiao Zhang ◽  
Xi-Mei Xue ◽  
Yu Yan ◽  
Jun Ye
Keyword(s):  
Sinorhizobium Meliloti ◽  
Size Exclusion ◽  
Diffusion Method ◽  
Flavin Mononucleotide ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Exclusion Chromatography ◽  
Pcc 6803

ArsH is an NADPH-dependent flavin mononucleotide reductase and is frequently encoded as part of anarsoperon. The function of thearsHgene remains to be characterized. Crystallization and structural studies may contribute to elucidating the specific biological function of ArsH associated with arsenic resistance. ArsH fromSynechocystissp. strain PCC 6803 was overproduced, purified and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion method. Diffraction data were collected and processed to a resolution of 1.6 Å. The crystals belonged to the tetragonal space groupI4122, with unit-cell parametersa=b= 127.94,c= 65.86 Å and one molecule in the asymmetric unit. Size-exclusion chromatography and molecular-replacement results showed that the ArsH formed a tetramer. Further structural analysis and comparison with ArsH fromSinorhizobium melilotiwill provide information about the oligomerization of ArsH.

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 Purification, crystallization and preliminary X-ray diffraction analysis of NADP-dependent glutamate dehydrogenase fromAspergillus niger

2014 ◽  
Vol 70 (11) ◽  
pp. 1508-1512 ◽  
Author(s):  
Prem Prakash ◽  
Adhish S. Walvekar ◽  
Narayan S. Punekar ◽  
Prasenjit Bhaumik
Keyword(s):  
Glutamate Dehydrogenase ◽  
Model Building ◽  
Specific Activity ◽  
Structure Refinement ◽  
High Specific Activity ◽  
Molecular Replacement ◽  
X Ray Diffraction ◽  
X Ray ◽  
Cell Parameters ◽  
Carbon And Nitrogen

Glutamate dehydrogenase (GDH) catalyzes the NAD-dependent or NADP-dependent oxidative deamination of L-glutamate to 2-oxoglutarate and ammonia. This important reversible reaction establishes the link between carbon and nitrogen metabolism. In this study,Aspergillus nigerNADP-GDH (AnGDH) has been overexpressed and purified. Purified AnGDH, with a high specific activity of 631.1 units per milligram of protein, was crystallized and the crystal diffracted to 2.9 Å resolution using a home X-ray source. Preliminary analysis of the X-ray diffraction data showed that the crystal belonged to space groupR32, with unit-cell parametersa=b= 173.8,c= 241.5 Å, α = β = 90, γ = 120°. The crystals exhibited an unusually high solvent content (83.0%) and had only one molecule in the asymmetric unit. Initial phases were obtained by molecular replacement, and model building and structure refinement of AnGDH are in progress.

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