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 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 Expression, purification and preliminary crystallographic analysis of the cryptic polo-box domain ofCaenorhabditis elegansZYG-1

2014 ◽  
Vol 70 (10) ◽  
pp. 1346-1350
Author(s):  
Ekaterina Shimanovskaya ◽  
Gang Dong
Keyword(s):  
Crystallographic Analysis ◽  
Structural Knowledge ◽  
Diffusion Method ◽  
Lysine Methylation ◽  
Wild Type ◽  
Data Set ◽  
Cell Parameters ◽  
Anomalous Data ◽  
Strain Bl21 ◽  
Shed Light

ZYG-1 is a polo-like kinase essential for centriole assembly inCaenorhabditis elegans. The targeting of ZYG-1 to nascent centrioles isviaits central cryptic polo-box (CPB) domain. To shed light on the molecular basis of ZYG-1 recruitment, it is necessary to obtain structural knowledge of the ZYG-1 CPB. Here, the expression, purification and preliminary crystallographic analysis of the ZYG-1 CPB are reported. The protein was overexpressed inEscherichia colistrain BL21 (DE3), purified by multi-step chromatography and crystallized using the vapour-diffusion method. Crystals of the wild-type protein exhibited an order–disorder pathology, which was solved by reductive lysine methylation. A complete anomalous data set was collected to 2.54 Å resolution at the Se Kedge (λ = 0.9792 Å). The crystal belonged to space groupP2, with unit-cell parametersa= 53.3,b= 60.09,c= 87.51 Å, β = 93.31°. There were two molecules in the asymmetric unit.

scholarly journals Cloning, purification and preliminary crystallographic analysis of Ara127N, a GH127 β-L-arabinofuranosidase fromGeobacillus stearothermophilusT6

2014 ◽  
Vol 70 (8) ◽  
pp. 1038-1045 ◽  
Author(s):  
Shifra Lansky ◽  
Rachel Salama ◽  
Roie Dann ◽  
Izhak Shner ◽  
Babu A. Manjasetty ◽  
...  
Keyword(s):  
Diffraction Data ◽  
Bifidobacterium Longum ◽  
Unit Cell ◽  
Monoclinic Space Group ◽  
Wild Type ◽  
Unit Cell Parameters ◽  
Data Set ◽  
Space Group ◽  
Cell Parameters ◽  
Transcriptional Units

The L-arabinan utilization system ofGeobacillus stearothermophilusT6 is composed of five transcriptional units that are clustered within a 38 kb DNA segment. One of the transcriptional units contains 11 genes, the last gene of which (araN) encodes a protein, Ara127N, that belongs to the newly established GH127 family. Ara127N shares 44% sequence identity with the recently characterized HypBA1 protein fromBifidobacterium longumand thus is likely to function similarly as a β-L-arabinofuranosidase. β-L-Arabinofuranosidases are enzymes that hydrolyze β-L-arabinofuranoside linkages, the less common form of such linkages, a unique enzymatic activity that has been identified only recently. The interest in the structure and mode of action of Ara127N therefore stems from its special catalytic activity as well as its membership of the new GH127 family, the structure and mechanism of which are only starting to be resolved. Ara127N has recently been cloned, overexpressed, purified and crystallized. Two suitable crystal forms have been obtained: one (CTP form) belongs to the monoclinic space groupP21, with unit-cell parametersa= 104.0,b= 131.2,c= 107.6 Å, β = 112.0°, and the other (RB form) belongs to the orthorhombic space groupP212121, with unit-cell parametersa= 65.5,b= 118.1,c= 175.0 Å. A complete X-ray diffraction data set has been collected to 2.3 Å resolution from flash-cooled crystals of the wild-type enzyme (RB form) at −173°C using synchrotron radiation. A selenomethionine derivative of Ara127N has also been prepared and crystallized for multi-wavelength anomalous diffraction (MAD) experiments. Crystals of selenomethionine Ara127N appeared to be isomorphous to those of the wild type (CTP form) and enabled the measurement of a three-wavelength MAD diffraction data set at the selenium absorption edge. These data are currently being used for detailed three-dimensional structure determination of the Ara127N protein.

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.

Substitution of murine ferrochelatase glutamate-287 with glutamine or alanine leads to porphyrin substrate-bound variants

2001 ◽  
Vol 356 (1) ◽  
pp. 217-222 ◽  
Author(s):  
Ricardo FRANCO ◽  
Alice S. PEREIRA ◽  
Pedro TAVARES ◽  
Arianna MANGRAVITA ◽  
Michael J. BARBER ◽  
...  
Keyword(s):  
Absorption Spectra ◽  
Catalytic Mechanism ◽  
Protoporphyrin Ix ◽  
Three Dimensional ◽  
Iron Chelation ◽  
Enzymic Activity ◽  
Dimensional Structure ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Flow Experiments

Ferrochelatase (EC 4.99.1.1) is the terminal enzyme of the haem biosynthetic pathway and catalyses iron chelation into the protoporphyrin IX ring. Glutamate-287 (E287) of murine mature ferrochelatase is a conserved residue in all known sequences of ferrochelatase, is present at the active site of the enzyme, as inferred from the Bacillus subtilis ferrochelatase three-dimensional structure, and is critical for enzyme activity. Substitution of E287 with either glutamine (Q) or alanine (A) yielded variants with lower enzymic activity than that of the wild-type ferrochelatase and with different absorption spectra from the wild-type enzyme. In contrast to the wild-type enzyme, the absorption spectra of the variants indicate that these enzymes, as purified, contain protoporphyrin IX. Identification and quantification of the porphyrin bound to the E287-directed variants indicate that approx. 80% of the total porphyrin corresponds to protoporphyrin IX. Significantly, rapid stopped-flow experiments of the E287A and E287Q variants demonstrate that reaction with Zn2+ results in the formation of bound Zn-protoporphyrin IX, indicating that the endogenously bound protoporphyrin IX can be used as a substrate. Taken together, these findings suggest that the structural strain imposed by ferrochelatase on the porphyrin substrate as a critical step in the enzyme catalytic mechanism is also accomplished by the E287A and E287Q variants, but without the release of the product. Thus E287 in murine ferrochelatase appears to be critical for the catalytic process by controlling the release of the product.

scholarly journals Solvent Accessibility of Residues Undergoing Pathogenic Variations in Humans: From Protein Structures to Protein Sequences

2021 ◽  
Vol 7 ◽  
Author(s):  
Castrense Savojardo ◽  
Matteo Manfredi ◽  
Pier Luigi Martelli ◽  
Rita Casadio
Keyword(s):  
Solvent Accessibility ◽  
Protein Structures ◽  
Three Dimensional ◽  
Protein Sequences ◽  
Large Data ◽  
Human Protein ◽  
Dimensional Structure ◽  
Wild Type ◽  
Solvent Exposure ◽  
Data Set

Solvent accessibility (SASA) is a key feature of proteins for determining their folding and stability. SASA is computed from protein structures with different algorithms, and from protein sequences with machine-learning based approaches trained on solved structures. Here we ask the question as to which extent solvent exposure of residues can be associated to the pathogenicity of the variation. By this, SASA of the wild-type residue acquires a role in the context of functional annotation of protein single-residue variations (SRVs). By mapping variations on a curated database of human protein structures, we found that residues targeted by disease related SRVs are less accessible to solvent than residues involved in polymorphisms. The disease association is not evenly distributed among the different residue types: SRVs targeting glycine, tryptophan, tyrosine, and cysteine are more frequently disease associated than others. For all residues, the proportion of disease related SRVs largely increases when the wild-type residue is buried and decreases when it is exposed. The extent of the increase depends on the residue type. With the aid of an in house developed predictor, based on a deep learning procedure and performing at the state-of-the-art, we are able to confirm the above tendency by analyzing a large data set of residues subjected to variations and occurring in some 12,494 human protein sequences still lacking three-dimensional structure (derived from HUMSAVAR). Our data support the notion that surface accessible area is a distinguished property of residues that undergo variation and that pathogenicity is more frequently associated to the buried property than to the exposed one.

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 Purification, crystallization and preliminary crystallographic analysis of a 4-thiouridine synthetase–RNA complex

2013 ◽  
Vol 69 (4) ◽  
pp. 421-424 ◽  
Author(s):  
Peter-Thomas Naumann ◽  
Charles T. Lauhon ◽  
Ralf Ficner
Keyword(s):  
Diffraction Data ◽  
Thermotoga Maritima ◽  
Crystallographic Analysis ◽  
X Ray Diffraction ◽  
Unit Cell Parameters ◽  
Orthorhombic Space Group ◽  
X Ray ◽  
Cell Parameters ◽  
Dependent Modification ◽  
Rna Complex

The sulfurtransferase 4-thiouridine synthetase (ThiI) is involved in the ATP-dependent modification of U8 in tRNA. ThiI fromThermotoga maritimawas cloned, overexpressed and purified. A complex comprising ThiI and a truncated tRNA was prepared and crystallized, and X-ray diffraction data were collected to a resolution of 3.5 Å. The crystals belonged to the orthorhombic space groupP212121, with unit-cell parametersa= 102.9,b= 112.8,c= 132.8 Å.

scholarly journals Ribokinase fromLeishmania donovani: purification, characterization and X-ray crystallographic analysis

2018 ◽  
Vol 74 (2) ◽  
pp. 99-104 ◽  
Author(s):  
Santhosh Gatreddi ◽  
Sayanna Are ◽  
Insaf Ahmed Qureshi
Keyword(s):  
Functional Characterization ◽  
Three Dimensional ◽  
Protozoan Parasite ◽  
Crystallographic Analysis ◽  
Size Exclusion ◽  
Dimensional Structure ◽  
Diffusion Method ◽  
Gene Encoding ◽  
Cell Parameters ◽  
Α Helix

Leishmaniais an auxotrophic protozoan parasite which acquires D-ribose by transporting it from the host cell and also by the hydrolysis of nucleosides. The enzyme ribokinase (RK) catalyzes the first step of ribose metabolism by phosphorylating D-ribose using ATP to produce D-ribose-5-phosphate. To understand its structure and function, the gene encoding RK fromL. donovaniwas cloned, expressed and purified using affinity and size-exclusion chromatography. Circular-dichroism spectroscopy of the purified protein showed comparatively more α-helix in the secondary-structure content, and thermal unfolding revealed theTmto be 317.2 K. Kinetic parameters were obtained by functional characterization ofL. donovaniRK, and theKmvalues for ribose and ATP were found to be 296 ± 36 and 116 ± 9.0 µM, respectively. Crystals obtained by the hanging-drop vapour-diffusion method diffracted to 1.95 Å resolution and belonged to the hexagonal space groupP61, with unit-cell parametersa=b= 100.25,c= 126.77 Å. Analysis of the crystal content indicated the presence of two protomers in the asymmetric unit, with a Matthews coefficient (VM) of 2.45 Å3 Da−1and 49.8% solvent content. Further study revealed that human counterpart of this protein could be used as a template to determine the first three-dimensional structure of the RK from trypanosomatid parasites.

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.

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