scholarly journals Purification, crystallization and preliminary crystallographic analysis of Gan1D, a GH1 6-phospho-β-galactosidase fromGeobacillus stearothermophilusT1

2014 ◽  
Vol 70 (2) ◽  
pp. 225-231 ◽  
Author(s):  
Shifra Lansky ◽  
Arie Zehavi ◽  
Roie Dann ◽  
Hay Dvir ◽  
Hassan Belrhali ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Extracellular Enzymes ◽  
Biochemical Characterization ◽  
Crystal Structure Analysis ◽  
Crystallographic Analysis ◽  
Glycoside Hydrolases ◽  
Cell Wall Polysaccharides ◽  
Data Set ◽  
Cell Parameters ◽  
Wide Range

Geobacillus stearothermophilusT1 is a Gram-positive thermophilic soil bacterium that contains an extensive system for the utilization of plant cell-wall polysaccharides, including xylan, arabinan and galactan. The bacterium uses a number of extracellular enzymes that break down the high-molecular-weight polysaccharides into short oligosaccharides, which enter the cell and are further hydrolyzed into sugar monomers by dedicated intracellular glycoside hydrolases. The interest in the biochemical characterization and structural analysis of these proteins originates mainly from the wide range of their potential biotechnological applications. Studying the different hemicellulolytic utilization systems inG. stearothermophilusT1, a new galactan-utilization gene cluster was recently identified, which encodes a number of proteins, one of which is a GH1 putative 6-phospho-β-galactosidase (Gan1D). Gan1D has recently been cloned, overexpressed, purified and crystallized as part of its comprehensive structure–function study. The best crystals obtained for this enzyme belonged to the triclinic space groupP1, with average crystallographic unit-cell parameters ofa = 67.0,b= 78.1,c= 92.1 Å, α = 102.4, β = 93.5, γ = 91.7°. A full diffraction data set to 1.33 Å resolution has been collected for the wild-type enzyme, as measured from flash-cooled crystals at 100 K, using synchrotron radiation. These data are currently being used for the detailed three-dimensional crystal structure analysis of Gan1D.

scholarly journals Preliminary crystallographic analysis of a double mutant of the acetyl xylo-oligosaccharide esterase Axe2 in its dimeric form

2014 ◽  
Vol 70 (4) ◽  
pp. 476-481 ◽  
Author(s):  
Shifra Lansky ◽  
Onit Alalouf ◽  
Rachel Salama ◽  
Hay Dvir ◽  
Yuval Shoham ◽  
...  
Keyword(s):  
Plant Cell Wall ◽  
Plant Biomass ◽  
Thermophilic Bacterium ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Orthorhombic Space Group ◽  
Data Set ◽  
Cell Parameters ◽  
Wide Range ◽  
Dimeric Form

Xylans are polymeric sugars constituting a significant part of the plant cell wall. They are usually substituted with acetyl side groups attached at positions 2 or 3 of the xylose backbone units. Acetylxylan esterases are part of the hemicellulolytic system of many microorganisms which utilize plant biomass for growth. These enzymes hydrolyze the ester linkages of the xylan acetyl groups and thus improve the accessibility of main-chain-hydrolyzing enzymes and their ability to break down the sugar backbone units. The acetylxylan esterases are therefore critically important for those microorganisms and as such could be used for a wide range of biotechnological applications. The structure of an acetylxylan esterase (Axe2) isolated from the thermophilic bacteriumGeobacillus stearothermophilusT6 has been determined, and it has been demonstrated that the wild-type enzyme is present as a unique torus-shaped octamer in the crystal and in solution. In order to understand the functional origin of this unique oligomeric structure, a series of rational noncatalytic, site-specific mutations have been made on Axe2. Some of these mutations led to a different dimeric form of the protein, which showed a significant reduction in catalytic activity. One of these double mutants, Axe2-Y184F-W190P, has recently been overexpressed, purified and crystallized. The best crystals obtained belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 71.1,b= 106.0,c= 378.6 Å. A full diffraction data set to 2.3 Å resolution has been collected from a flash-cooled crystal of this type at 100 K using synchrotron radiation. This data set is currently being used for the three-dimensional structure analysis of the Axe2-Y184F-W190P mutant in its dimeric form.

scholarly journals Crystallization and preliminary crystallographic analysis of Axe2, an acetylxylan esterase fromGeobacillus stearothermophilus

2013 ◽  
Vol 69 (4) ◽  
pp. 430-434 ◽  
Author(s):  
Shifra Lansky ◽  
Onit Alalouf ◽  
Vered Solomon ◽  
Anat Alhassid ◽  
Lata Govada ◽  
...  
Keyword(s):  
Diffraction Data ◽  
Biochemical Characterization ◽  
Plant Biomass ◽  
Crystallographic Analysis ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Data Set ◽  
Cell Parameters ◽  
Acetylxylan Esterase

Acetylxylan esterases are part of the hemi-cellulolytic system of many microorganisms which utilize plant biomass for growth. Xylans, which are polymeric sugars that constitute a significant part of the plant biomass, are usually substituted with acetyl side groups attached at position 2 or 3 of the xylose backbone units. Acetylxylan esterases hydrolyse the ester linkages of the xylan acetyl groups and thus improve the ability of main-chain hydrolysing enzymes to break down the sugar backbone units. As such, these enzymes play an important part in the hemi-cellulolytic utilization system of many microorganisms that use plant biomass for growth. Interest in the biochemical characterization and structural analysis of these enzymes stems from their numerous potential biotechnological applications. An acetylxylan esterase (Axe2) of this type fromGeobacillus stearothermophilusT-6 has recently been cloned, overexpressed, purified, biochemically characterized and crystallized. One of the crystal forms obtained (RB1) belonged to the tetragonal space groupI422, with unit-cell parametersa=b= 110.2,c= 213.1 Å. A full diffraction data set was collected to 1.85 Å resolution from flash-cooled crystals of the wild-type enzyme at 100 K using synchrotron radiation. A selenomethionine derivative of Axe2 has also been prepared and crystallized for single-wavelength anomalous diffraction experiments. The crystals of the selenomethionine-derivatized Axe2 appeared to be isomorphous to those of the wild-type enzyme and enabled the measurement of a full 1.85 Å resolution diffraction data set at the selenium absorption edge and a full 1.70 Å resolution data set at a remote wavelength. These data are currently being used for three-dimensional structure determination of the Axe2 protein.

scholarly journals Functional Analyses of Multiple Lichenin-Degrading Enzymes from the Rumen Bacterium Ruminococcus albus 8

2011 ◽  
Vol 77 (21) ◽  
pp. 7541-7550 ◽  
Author(s):  
Michael Iakiviak ◽  
Roderick I. Mackie ◽  
Isaac K. O. Cann
Keyword(s):  
Plant Cell Wall ◽  
Bioinformatic Analysis ◽  
Major Product ◽  
Glycoside Hydrolases ◽  
Rumen Bacterium ◽  
Cell Wall Polysaccharides ◽  
Ruminococcus Albus ◽  
Content Type ◽  
Wide Range ◽  
Functional Analyses

ABSTRACTRuminococcus albus8 is a fibrolytic ruminal bacterium capable of utilization of various plant cell wall polysaccharides. A bioinformatic analysis of a partial genome sequence ofR. albusrevealed several putative enzymes likely to hydrolyze glucans, including lichenin, a mixed-linkage polysaccharide of glucose linked together in β-1,3 and β-1,4 glycosidic bonds. In the present study, we demonstrate the capacity of four glycoside hydrolases (GHs), derived fromR. albus, to hydrolyze lichenin. Two of the genes encoded GH family 5 enzymes (Ra0453 and Ra2830), one gene encoded a GH family 16 enzyme (Ra0505), and the last gene encoded a GH family 3 enzyme (Ra1595). Each gene was expressed inEscherichia coli, and the recombinant protein was purified to near homogeneity. Upon screening on a wide range of substrates, Ra0453, Ra2830, and Ra0505 displayed different hydrolytic properties, as they released unique product profiles. The Ra1595 protein, predicted to function as a β-glucosidase, preferred cleavage of a nonreducing end glucose when linked by a β-1,3 glycosidic bond to the next glucose residue. The major product of Ra0505 hydrolysis of lichenin was predicted to be a glucotriose that was degraded only by Ra0453 to glucose and cellobiose. Most importantly, the four enzymes functioned synergistically to hydrolyze lichenin to glucose, cellobiose, and cellotriose. This lichenin-degrading enzyme mix should be of utility as an additive to feeds administered to monogastric animals, especially those high in fiber.

scholarly journals YopT domain of the PfhB2 toxin from Pasteurella multocida: protein expression, characterization, crystallization and crystallographic analysis

2018 ◽  
Vol 74 (3) ◽  
pp. 128-134
Author(s):  
Sanjeev Kumar ◽  
Victoria Hedrick ◽  
Seema Mattoo
Keyword(s):  
Pasteurella Multocida ◽  
Opportunistic Infections ◽  
Structural Information ◽  
Sequence Similarity ◽  
Catalytic Triad ◽  
Crystallographic Analysis ◽  
High Sequence Similarity ◽  
Data Set ◽  
Cell Parameters ◽  
Tract Infections

Pasteurella multocida causes respiratory-tract infections in a broad range of animals, as well as opportunistic infections in humans. P. multocida secretes a multidomain toxin called PfhB2, which contains a YopT-like cysteine protease domain at its C-terminus. The YopT domain of PfhB2 contains a well conserved Cys–His–Asp catalytic triad that defines YopT family members, and shares high sequence similarity with the prototype YopT from Yersinia sp. To date, only one crystal structure of a YopT family member has been reported; however, additional structural information is needed to help characterize the varied substrate specificity and enzymatic action of this large protease family. Here, a catalytically inactive C3733S mutant of PfhB2 YopT that provides enhanced protein stability was used with the aim of gaining structural insight into the diversity within the YopT protein family. To this end, the C3733S mutant of PfhB2 YopT has been successfully cloned, overexpressed, purified and crystallized. Diffraction data sets were collected from native crystals to 3.5 Å resolution and a single-wavelength anomalous data set was collected from an iodide-derivative crystal to 3.2 Å resolution. Data pertaining to crystals belonging to space group P31, with unit-cell parameters a = 136.9, b = 136.9, c = 74.7 Å for the native crystals and a = 139.2, b = 139.2, c = 74.7 Å for the iodide-derivative crystals, are discussed.

scholarly journals Production, crystallization and preliminary crystallographic analysis ofAllochromatium vinosumthiosulfate dehydrogenase TsdA, an unusual acidophilicc-type cytochrome

2014 ◽  
Vol 70 (10) ◽  
pp. 1424-1427 ◽  
Author(s):  
José A. Brito ◽  
André Gutierres ◽  
Kevin Denkmann ◽  
Christiane Dahl ◽  
Margarida Archer
Keyword(s):  
Crystallographic Analysis ◽  
Sulfur Cycle ◽  
Monoclinic Space Group ◽  
Asymmetric Unit ◽  
Oxidative Condensation ◽  
Unit Cell Parameters ◽  
Allochromatium Vinosum ◽  
Data Set ◽  
Purple Sulfur Bacterium ◽  
Cell Parameters

The ability to perform the very simple oxidation of two molecules of thiosulfate to tetrathionate is widespread among prokaryotes. Despite the prevalent occurrence of tetrathionate formation and its well documented significance within the sulfur cycle, little is known about the enzymes that catalyze the oxidative condensation of two thiosulfate anions. To fill this gap, the thiosulfate dehydrogenase (TsdA) enzyme from the purple sulfur bacteriumAllochromatium vinosumwas recombinantly expressed inEscherichia coli, purified and crystallized, and a crystallographic data set was collected. The crystals belonged to the monoclinic space groupC2, with unit-cell parametersa= 79.2,b= 69.9,c= 57.9 Å, β = 129.3°, contained one monomer per asymmetric unit and diffracted to a resolution of 1.98 Å.

scholarly journals Expression, purification, crystallization and preliminary X-ray crystallographic analysis of Enpp6

2014 ◽  
Vol 70 (6) ◽  
pp. 794-799 ◽  
Author(s):  
Junko Morita ◽  
Kazuki Kato ◽  
Emiko Mihara ◽  
Ryuichiro Ishitani ◽  
Junichi Takagi ◽  
...  
Keyword(s):  
Catalytic Domain ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Choline Metabolism ◽  
Protein Molecules ◽  
Membrane Bound

Enpp (ectonucleotide phosphodiesterase/pyrophosphatase) 6 is a membrane-bound glycoprotein that hydrolyzes choline-containing compounds such as lysophosphatidylcholine and glycerophosphorylcholine, and presumably participates in choline metabolism. The catalytic domain of mouse Enpp6 was expressed in HEK293T cells, purified using the TARGET tag/P20.1-Sepharose system and crystallized. An X-ray diffraction data set was collected to 1.8 Å resolution. The crystal belonged to space groupP1, with unit-cell parametersa= 63.7,b= 68.8,c= 69.7 Å, α = 60.6, β = 87.0, γ = 68.1°. Assuming the presence of two protein molecules per asymmetric unit, the solvent content was estimated to be 49.5%.

scholarly journals Cloning, purification and preliminary crystallographic analysis of theHelicobacter pyloripseudaminic acid biosynthesisN-acetyltransferase PseH

2014 ◽  
Vol 70 (9) ◽  
pp. 1276-1279 ◽  
Author(s):  
Yu C. Liu ◽  
Abu I. Ud-Din ◽  
Anna Roujeinikova
Keyword(s):  
Crystallographic Analysis ◽  
Diffusion Method ◽  
X Ray Diffraction ◽  
Data Set ◽  
Cell Parameters ◽  
Nonulosonic Acid ◽  
Pseudaminic Acid ◽  
The Common ◽  
H Pylori ◽  
Development Of Gastric Cancer

Helicobacter pyloriinfection is the common cause of gastritis and duodenal and stomach ulcers, which have been linked to a higher risk of the development of gastric cancer. The motility that facilitates persistent infection requires functional flagella that are heavily glycosylated with 5,7-diacetamido-3,5,7,9-tetradeoxy-L-glycero-L-manno-nonulosonic acid (pseudaminic acid). Pseudaminic acid biosynthesis protein H (PseH) catalyzes the third step in its biosynthetic pathway, producing UDP-2,4-diacetamido-2,4,6-trideoxy-β-L-altropyranose. Crystals ofH. pyloriPseH have been grown by the hanging-drop vapour-diffusion method using diammonium tartrate as a precipitating agent. The crystals belonged to space groupI222 orI212121, with unit-cell parametersa= 107.8,b= 145.4,c= 166.3 Å. A complete X-ray diffraction data set has been collected to 2.5 Å resolution using cryocooling conditions and synchrotron radiation.

scholarly journals Cloning, expression, purification, crystallization and preliminary crystallographic analysis of the C-terminal domain of Par-4 (PAWR)

2014 ◽  
Vol 70 (9) ◽  
pp. 1224-1227 ◽  
Author(s):  
Udaya Kumar Tiruttani Subhramanyam ◽  
Jan Kubicek ◽  
Ulf B. Eidhoff ◽  
Joerg Labahn
Keyword(s):  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Data Set ◽  
X Ray ◽  
Cell Parameters ◽  
Intrinsically Disordered ◽  
Tumour Suppressor Function ◽  
C Terminus ◽  
Apoptotic Protein

Prostate apoptosis response-4 protein is an intrinsically disordered pro-apoptotic protein with tumour suppressor function. Par-4 is known for its selective induction of apoptosis in cancer cells only and its ability to interact with various apoptotic proteinsviaits C-terminus. Par-4, with its unique function and various interacting partners, has gained importance as a potential target for cancer therapy. The C-terminus of the rat homologue of Par-4 was crystallized and a 3.7 Å resolution X-ray diffraction data set was collected. Preliminary data analysis shows the space group to beP41212. The unit-cell parameters area=b= 115.351,c= 123.663 Å, α = β = γ = 90°.

scholarly journals Crystallographic analysis of the Staphylococcus epidermidis lipase involved in esterification in aqueous solution

2018 ◽  
Vol 74 (6) ◽  
pp. 351-354
Author(s):  
Cheng-Huan Liu ◽  
Yu-Ting Chen ◽  
Ming-Hon Hou ◽  
Nien-Jen Hu ◽  
Chin-Shuh Chen ◽  
...  
Keyword(s):  
Aqueous Solution ◽  
Molecular Weight ◽  
Staphylococcus Epidermidis ◽  
Crystallographic Analysis ◽  
Asymmetric Unit ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Solvent Content ◽  
Cell Parameters ◽  
Esterification In Aqueous Solution

The Staphylococcus epidermidis lipase (SeLip, GehC) can be used in flavour-compound production via esterification in aqueous solution. This study reports the crystallization and crystallographic analysis of recombinant GehC (rGehC; Lys303–Lys688) with a molecular weight of 43 kDa. rGehC was crystallized at 293 K using PEG 10 000 as a precipitant, and a 99.9% complete native data set was collected from a cooled crystal at 77 K to a resolution of 1.9 Å with an overall R merge value of 7.3%. The crystals were orthorhombic and belonged to space group P212121, with unit-cell parameters a = 42.07, b = 59.31, c = 171.30 Å, α = β = γ = 90°. Solvent-content calculations suggest that there is likely to be one lipase subunit in the asymmetric unit.

scholarly journals Crystallization and preliminary X-ray crystallographic analysis of Pz peptidase B fromGeobacillus collagenovoransMO-1

2012 ◽  
Vol 68 (7) ◽  
pp. 757-759 ◽  
Author(s):  
Hiroaki Nakano ◽  
Allin Hosokawa ◽  
Ryuji Tagawa ◽  
Koji Inaka ◽  
Kazunori Ohta ◽  
...  
Keyword(s):  
Space Station ◽  
Crystallographic Analysis ◽  
Diffusion Method ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Peptide Substrates ◽  
X Ray ◽  
Cell Parameters ◽  
Experiment Module ◽  
Counter Diffusion

Pz peptidase B is an intracellular M3 metallopeptidase that is found together with Pz peptidase A in the thermophileGeobacillus collagenovoransMO-1 and recognizes collagen-specific tripeptide units (-Gly-Pro-X-). These peptidases have low homology in their primary structures; however, their cleavage patterns towards peptide substrates are similar. In this work, Pz peptidase B was crystallized using the counter-diffusion method. Data were collected to a resolution of 1.6 Å at 100 K from a crystal obtained in the Japanese Experiment Module (JEM; also known as `Kibo') at the International Space Station (ISS). The crystal belonged to the trigonal space groupP3121, with unit-cell parametersa=b= 87.64,c= 210.5 Å. A complete data set was also obtained from crystals of selenomethionine-substituted protein.

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