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 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 Crystallization and preliminary X-ray diffraction analysis of a novel β-L-arabinofuranosidase (HypBA1) fromBifidobacterium longum

2014 ◽  
Vol 70 (5) ◽  
pp. 636-638 ◽  
Author(s):  
Zhen Zhu ◽  
Miao He ◽  
Chun-Hsiang Huang ◽  
Tzu-Ping Ko ◽  
Yi-Fang Zeng ◽  
...  
Keyword(s):  
Glycoside Hydrolase ◽  
Bifidobacterium Longum ◽  
Data Bank ◽  
Diffusion Method ◽  
Glycoside Hydrolase Family ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
X Ray ◽  
Cell Parameters ◽  
Hydrolase Family

The β-L-arabinofuranosidase (HypBA1) fromBifidobacterium longumJCM 1217 hydrolyzes the β-1,2-linked arabinofuranose disaccharide to release L-arabinoses. HypBA1 was classified into glycoside hydrolase family 127 (GH127) by the CAZy website (http://www.cazy.org/). The enzyme was expressed inEscherichia coliand the purified recombinant protein was crystallized. Crystals belonging to the primitive hexagonal space groupP3x21, with unit-cell parametersa=b= 75.9,c= 254.0 Å, were obtained by the sitting-drop vapour-diffusion method and diffracted to 2.78 Å resolution. ABLASTPsearch (http://blast.ncbi.nlm.nih.gov/) of the Protein Data Bank did not reveal any similar crystal structures. Structural determination by using SeMet MAD and MIR methods is in progress.

scholarly journals Epoxyalkyl glycosides of d-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10

2000 ◽  
Vol 347 (3) ◽  
pp. 865-873 ◽  
Author(s):  
Patricia NTARIMA ◽  
Wim NERINCKX ◽  
Klaus KLARSKOV ◽  
Bart DEVREESE ◽  
Mahalingeshwara K. BHAT ◽  
...  
Keyword(s):  
Active Site ◽  
Glycoside Hydrolase ◽  
Glycoside Hydrolases ◽  
Thermomyces Lanuginosus ◽  
Glycoside Hydrolase Family ◽  
Active Site Residues ◽  
X Ray ◽  
Order Of Magnitude ◽  
Site Markers ◽  
Hydrolase Family

A series of Ω-epoxyalkyl glycosides of D-xylopyranose, xylobiose and xylotriose were tested as potential active-site-directed inhibitors of xylanases from glycoside hydrolase families 10 and 11. Whereas family-10 enzymes (Thermoascus aurantiacus Xyn and Clostridium thermocellum Xyn Z) are resistant to electrophilic attack of active-site carboxyl residues, glycoside hydrolases of family 11 (Thermomyces lanuginosus Xyn and Trichoderma reesei Xyn II) are irreversibly inhibited. The apparent inactivation and association constants (ki, 1/Ki) are one order of magnitude higher for the xylobiose and xylotriose derivatives. The effects of the aglycone chain length can clearly be described. Xylobiose and n-alkyl β-D-xylopyranosides are competitive ligands and provide protection against inactivation. MS measurements showed 1:1 stoichiometries in most labelling experiments. Electrospray ionization MS/MS analysis revealed the nucleophile Glu86 as the modified residue in the T. lanuginosus xylanase when 2,3-epoxypropyl β-D-xylopyranoside was used, whereas the acid/base catalyst Glu178 was modified by the 3,4-epoxybutyl derivative. The active-site residues Glu86 and Glu177 in T. reesei Xyn II are similarly modified, confirming earlier X-ray crystallographic data [Havukainen, Törrönen, Laitinen and Rouvinen (1996) Biochemistry 35, 9617-9624]. The inability of the Ω-epoxyalkyl xylo(oligo)saccharide derivatives to inactivate family-10 enzymes is discussed in terms of different ligand-subsite interactions.

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.

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.

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 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.

Sign in / Sign up

Export Citation Format

Share Document