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1.  Expression, purification, crystallization and preliminary X-ray crystallographic analysis of pantothenate kinase from Mycobacterium tuberculosis  
Pantothenate kinase, the first enzyme of the universal coenzyme A biosynthetic pathway, from M. tuberculosis H37Rv has been cloned, expressed, purified and X-ray analysed in two different crystal forms.
Pantothenate kinase is an essential enzyme in the bacterial life cycle. It catalyzes the phosphorylation of pantothenate (vitamin B5) to 4′-phosphopantothenate, the first step in the coenzyme A biosynthetic pathway. The enzyme from Mycobacterium tuberculosis, MW 35.7 kDa, has been cloned, expressed, purified and crystallized in two different trigonal crystal forms, both belonging to space group P3121. Two complete data sets of resolution 2.5 Å (form I) and 2.9 Å (form II) from crystals with unit-cell parameters a = b = 78.3, c = 115.45 Å and a = b = 107.63, c = 89.85 Å, respectively, were collected at room temperature on a home X-ray source. Structures of both crystal forms were solved for one subunit in the asymmetric unit by molecular replacement.
doi:10.1107/S1744309104028040
PMCID: PMC1952411  PMID: 16508093
pantothenate kinase
2.  Mistletoe lectin I in complex with galactose and lactose reveals distinct sugar-binding properties 
The structures of mistletoe lectin I in complex with lactose and galactose reveal differences in binding by the two known sites in subdomains α1 and γ2 and suggest the presence of a third low-affinity site in subdomain β1.
The structures of mistletoe lectin I (ML-I) from Viscum album complexed with lactose and galactose have been determined at 2.3 Å resolution and refined to R factors of 20.9% (R free = 23.6%) and 20.9 (R free = 24.6%), respectively. ML-I is a heterodimer and belongs to the class of ribosome-inactivating proteins of type II, which consist of two chains. The A-chain has rRNA N-glycosidase activity and irreversibly inhibits eukaryotic ribosomes. The B-chain is a lectin and preferentially binds to galactose-terminated glycolipids and glycoproteins on cell membranes. Saccharide binding is performed by two binding sites in subdomains α1 and γ2 of the ML-I B-chain separated by ∼62 Å from each other. The favoured binding of galactose in subdomain α1 is achieved via hydrogen bonds connecting the 4-hydroxyl and 3-hydroxyl groups of the sugar moiety with the side chains of Asp23B, Gln36B and Lys41B and the main chain of 26B. The aromatic ring of Trp38B on top of the preferred binding pocket supports van der Waals packing of the apolar face of galactose and stabilizes the sugar–lectin complex. In the galactose-binding site II of subdomain γ2, Tyr249B provides the hydrophobic stacking and the side chains of Asp235B, Gln238B and Asn256B are hydrogen-bonding partners for galactose. In the case of the galactose-binding site I, the 2-hydroxyl group also stabilizes the sugar–protein complex, an interaction thus far rarely detected in galactose-specific lectins. Finally, a potential third low-affinity galactose-binding site in subunit β1 was identified in the present ML-I structures, in which a glycerol molecule from the cryoprotectant buffer has bound, mimicking the sugar compound.
doi:10.1107/S1744309104031501
PMCID: PMC1952410  PMID: 16508080
ribosome-inactivation proteins; mistletoe lectin I; sugar-binding sites
3.  Cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of propionate kinase (TdcD) from Salmonella typhimurium  
Propionate kinase (TdcD) from S. typhimurium has been expressed, purified and crystallized. A diffraction data set has been collected to 2.2 Å resolution.
In the cell, propionate is mainly formed during β-oxidation of odd-numbered carbon-chain fatty acids, fermentation of carbohydrates and degradation of the amino acids threonine, valine, isoleucine and methionine. Recently, it has been shown that l-threonine is non-oxidatively cleaved to propionate via 2-­ketobutyrate. The last step in this process, conversion of propionyl phosphate and ADP to propionate and ATP, is catalysed by propionate kinase (EC 2.7.1.–). Here, the cloning of propionate kinase (molecular weight 44 kDa) from Salmonella typhimurium with an N-terminal hexahistidine affinity tag and its overexpression in Escherichia coli are reported. Purified propionate kinase was found to cocrystallize with ADP in the hanging-drop vapour-diffusion and microbatch methods. Crystals belong to space group P3121 or P3221, with unit-cell parameters a = b = 111.47, c = 66.52 Å. A complete data set to 2.2 Å resolution has been collected using an image-plate detector system mounted on a rotating-anode X-ray generator.
doi:10.1107/S1744309104026429
PMCID: PMC1952409  PMID: 16508089
TdcD; propionate kinases; acetate kinases; l-threonine metabolism
4.  Cloning, preparation and preliminary crystallographic studies of penicillin V acylase autoproteolytic processing mutants 
The production, crystallization and characterization of three inactive mutants of penicillin V acylase from B. sphaericus in their respective precursor and processed forms are reported. The space groups are different for the native enzyme and the mutants.
The crystallization of three catalytically inactive mutants of penicillin V acylase (PVA) from Bacillus sphaericus in precursor and processed forms is reported. The mutant proteins crystallize in different primitive monoclinic space groups that are distinct from the crystal forms for the native enzyme. Directed mutants and clone constructs were designed to study the post-translational autoproteolytic processing of PVA. The catalytically inactive mutants will provide three-dimensional structures of precursor PVA forms, plus open a route to the study of enzyme–substrate complexes for this industrially important enzyme.
doi:10.1107/S1744309104031227
PMCID: PMC1952408  PMID: 16508111
autoproteolysis; Ntn hydrolases; oxyanion holes; post-translational processing; precursor proteins; pro-peptides
5.  Preliminary crystallographic analysis of sugar cane phosphoribosylpyrophosphate synthase 
X-ray diffraction data have been collected from crystals of recombinant sugar cane phosphoribosylpyrophosphate synthase (PRS) and analysis has revealed its quaternary structure, localizing this PRS into the class of enzymes forming an hexameric oligomer of 223 kDa.
Phosphoribosylpyrophosphate synthases (PRS; EC 2.7.6.1) are enzymes that are of central importance in several metabolic pathways in all cells. The sugar cane PRS enzyme contains 328 amino acids with a molecular weight of 36.6 kDa and represents the first plant PRS to be crystallized, as well as the first phosphate-independent PRS to be studied in molecular detail. Sugar cane PRS was overexpressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. Using X-ray diffraction experiments it was determined that the crystals belong to the orthorhombic system, with space group P21212 and unit-cell parameters a = 213.2, b = 152.6, c = 149.3 Å. The crystals diffract to a maximum resolution of 3.3 Å and a complete data set to 3.5 Å resolution was collected and analysed.
doi:10.1107/S1744309104025825
PMCID: PMC1952407  PMID: 16508088
phosphoribosylpyrophosphate; PRPP synthase; sugar cane
6.  Crystallization and preliminary crystallographic study of a recombinant predicted acetamidase/formamidase from the thermophile Thermoanaerobacter tengcongensis  
A predicated acetamidase/formanidase from the archaeon T. tengcongensis and its SeMet substitute have been crystallized and undergone preliminarily crystallographic studies including MAD data collection.
No crystal structures are yet available for homologues of a predicted acetamidase/formamidase (Amds/Fmds) from the archaeon Thermoanaerobacter tengcongensis. The Amds/Fmds gene was cloned and expressed as a soluble protein in Escherichia coli. Native Amds/Fmds and its SeMet-substituted form were purified and crystallized by vapour diffusion in hanging drops at 296 K. The native crystals, which were grown in PEG 8000, belong to the monoclinic space group P21, with unit-cell parameters a = 41.23 (3), b = 152.88 (6), c = 100.26 (7) Å, β = 99.49 (3)°. The diffraction data were collected to 2.00 Å resolution using synchrotron radiation. Based on a predicted solvent content of 50%, a Matthews coefficient of 2.44 Å3 Da−1 and two main peaks in the self-rotation function, the asymmetric unit is predicted to contain two dimers of the 32 kDa native protein. MAD data were collected for the SeMet protein, but the corresponding crystals display different unit-cell parameters and appear to contain four dimers in the asymmetric unit.
doi:10.1107/S1744309104030519
PMCID: PMC1952406  PMID: 16508105
acetamidase/formamidase; Thermoanaerobacter tengcongensis
7.  Editorial 
doi:10.1107/S1744309104033834
PMCID: PMC1952405
Editorial
8.  Crystallization and preliminary X-ray characterization of a lectin from Cicer arietinum (chickpea) 
The crystallization and characterization of a lectin isolated and purified from C. arietinum and possessing complex sugar specificity is reported.
The lectin isolated from mature seeds of Cicer arietinum (CAL) agglutinates pronase-treated rabbit and human erythrocytes and its haemagglutination activity is inhibited by fetuin and desialated fetuin but not by simple monosaccharides or oligosaccharides. The purified lectin is a dimer of molecular weight 43 000 Da composed of two identical subunits (MW 21 500), as confirmed by SDS–PAGE. The lectin has been crystallized using the hanging-drop vapour-diffusion method at 295 K over a well solution containing 0.2 M sodium acetate, 0.1 M sodium phosphate buffer pH 6.5 and 14%(w/v) polyethylene glycol 8000. The triangular prism-shaped crystals belong to space group R3 and have unit-cell parameters a = b = 81.2, c = 69.4 Å. The diffraction data are 93.8% complete to 2.3 Å Bragg spacing with an R merge of 0.103.
doi:10.1107/S1744309104032166
PMCID: PMC1952404  PMID: 16508116
complex sugar specificity; legume lectin; seed albumins
9.  Overproduction, purification and preliminary X-ray diffraction analysis of a sulfotransferase from Mycobacterium tuberculosis H37Rv 
A sulfotransferase from M. tuberculosis was crystallized and preliminarily analyzed using X-ray diffraction.
Sulfotransferase STF1 from the Mycobacterium tuberculosis H37Rv genome was overproduced in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystals diffract to 1.5 Å resolution using synchrotron radiation at SPring-8. The crystals are monoclinic and belong to space group P21, with unit-cell parameters a = 40.86, b = 95.76, c = 48.04 Å, β = 106.43°. The calculated Matthews coefficient is approximately 2.1 Å3 Da−1 assuming the presence of one molecule of STF1 in the asymmetric unit. A substrate-binding assay using a PAP–agarose column suggests that STF1 exhibits sulfotransferase activity.
doi:10.1107/S1744309104022328
PMCID: PMC1952402  PMID: 16508083
sulfotransferase; Mycobacterium tuberculosis
10.  Crystallization and preliminary X-ray studies on the reaction center–light-harvesting 1 core complex from Rhodopseudomonas viridis  
The reaction center–light-harvesting 1 core complex from R. viridis was crystallized and X-ray diffraction data were collected to 8.0 Å resolution.
The reaction center–light-harvesting 1 (RC–LH1) core complex is the photosynthetic apparatus in the membrane of the purple photosynthetic bacterium Rhodopseudomonas viridis. The RC is surrounded by an LH1 complex that is constituted of oligomers of three types of apoproteins (α, β and γ chains) with associated bacteriochlorophyll bs and carotenoid. It has been crystallized by the sitting-drop vapour-diffusion method. A promising crystal diffracted to beyond 8.0 Å resolution. It belonged to space group P1, with unit-cell parameters a = 141.4, b = 136.9, c = 185.3 Å, α = 104.6, β = 94.0, γ = 110.7°. A Patterson function calculated using data between 15.0 and 8.0 Å resolution suggested that the LH1 complex is distributed with quasi-16-fold rotational symmetry around the RC.
doi:10.1107/S1744309104028945
PMCID: PMC1952401  PMID: 16508098
photosynthesis; reaction center–light-harvesting 1 core complex; Rhodopseudomonas viridis; integral membrane proteins
11.  On the purification and preliminary crystallographic analysis of isoquinoline 1-oxidoreductase from Brevundimonas diminuta 7 
Crystallization of isoquinoline 1-oxidoreductase from B. diminuta was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement.
Isoquinoline 1-oxidoreductase (IOR) from Brevundimonas diminuta is a mononuclear molybdoenzyme of the xanthine-dehydrogenase family of proteins and catalyzes the conversion of isoquinoline to isoquinoline-1-one. Its primary sequence and behaviour, specifically in its substrate specificity and lipophilicity, differ from other members of the family. A crystal structure of the enzyme is expected to provide an explanation for these differences. This paper describes the crystallization and preliminary X-ray diffraction experiments as well as an optimized purification protocol for IOR. Crystallization of IOR was achieved using two different crystallization buffers. Streak-seeding and cross-linking were essential to obtain well diffracting crystals. Suitable cryo-conditions were found and a structure solution was obtained by molecular replacement. However, phases need to be improved in order to obtain a more interpretable electron-density map.
doi:10.1107/S1744309104032105
PMCID: PMC1952400  PMID: 16508115
isoquinoline 1-oxidoreductase; xanthine oxidase/xanthine dehydrogenase; oxidoreductases; molybdenum enzymes; molybdopterin; Brevundimonas diminuta
12.  Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu 
Bacteriophage Mu baseplate protein gene product 44 was crystallized. The crystal belongs to space group R3, with unit-cell parameters a = b = 126.6, c = 64.2 Å.
Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 Å. The crystals diffract X-­rays to at least 2.1 Å resolution and are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 Å resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.
doi:10.1107/S1744309104029574
PMCID: PMC1952399  PMID: 16508104
gene product 44; bacteriophage Mu
13.  Crystallization and preliminary X-ray crystallographic analysis of MbtI, a protein essential for siderophore biosynthesis in Mycobacterium tuberculosis  
MbtI, the putative isochorismate synthase essential for siderophore biosynthesis in M. tuberculosis, has been crystallized. Diffraction data have been collected to 1.8 Å resolution.
Mycobacterium tuberculosis, the causative agent of tuberculosis, depends on the secretion of salicylate-based siderophores called mycobactins for the acquisition of extracellular iron, which is essential for the growth and virulence of the bacterium. The protein MbtI is thought to be the isochorismate synthase enzyme responsible for the conversion of chorismate to isochorismate, the first step in the salicylate production required for mycobactin biosynthesis. MbtI has been overexpressed in Escherichia coli, purified and crystallized. The crystals diffract to a maximum resolution of 1.8 Å. They belong to space group P212121, with unit-cell parameters a = 51.8, b = 163.4, c = 194.9 Å, consistent with the presence of either two, three or four molecules in the asymmetric unit.
doi:10.1107/S1744309104031215
PMCID: PMC1952396  PMID: 16508110
Mycobacterium tuberculosis; iron acquisition; siderophore biosynthesis
14.  Purification and crystallization of a trimodular complex comprising the type II cohesin–dockerin interaction from the cellulosome of Clostridium thermocellum  
A trimodular complex comprising the type II cohesin–dockerin interaction from the cellulosome of C. thermocellum has been purified and crystallized by the hanging-drop vapour-diffusion method. A native crystal and a selenomethionine derivative have been analyzed using X-ray diffraction.
The high-affinity calcium-mediated type II cohesin–dockerin interaction is responsible for the attachment of the multi-enzyme cellulose-degrading complex, termed the cellulosome, to the cell surface of the thermophilic anaerobe Clostridium thermocellum. A trimodular 40 kDa complex comprising the SdbA type II cohesin and the the CipA type II dockerin–X module modular pair from the cellulosome of C. thermocellum has been crystallized. The crystals belong to space group P212121, with unit-cell parameters a = 45.21, b = 52.34, c = 154.69 Å. The asymmetric unit contains one molecule of the protein complex and native and selenomethionine-derivative crystals diffracted to 2.1 and 2.0 Å, respectively.
doi:10.1107/S1744309104025837
PMCID: PMC1952395  PMID: 16508087
type II cohesin; dockerin; cellulosome
15.  Crystallization and preliminary X-ray diffraction studies of a catechol-O-methyltransferase/inhibitor complex 
Catechol-O-methyltransferase has been co-crystallized with a novel inhibitor, which has potential therapeutic application in the Parkinson’s disease therapy.
Inhibitors of the enzyme catechol-O-methyltransferase (COMT) are used as co-adjuvants in the therapy of Parkinson’s disease. A recombinant form of the soluble cytosolic COMT from rat has been co-crystallized with a new potent inhibitor, BIA 8-176 [(3,4-dihydroxy-2-nitrophenyl)phenylmethanone], by the vapour-diffusion method using PEG 6K as precipitant. Crystals diffract to 1.6 Å resolution on a synchrotron-radiation source and belong to the monoclinic space group P21, with unit-cell parameters a = 52.77, b = 79.63, c = 61.54 Å, β = 91.14°.
doi:10.1107/S1744309104031197
PMCID: PMC1952394  PMID: 16508109
catechol-O-methyltransferase; Parkinson’s disease; catechol-O-methyltransferase inhibitors
16.  Expression, purification, crystallization and preliminary crystallographic analysis of human Pim-­1 kinase 
Pim kinases, belong to a distinctive serine/threonine protein-kinase family and are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Human Pim-1 kinase has been cloned, expressed and crystallized
Pim kinases, including Pim-1, Pim-2 and Pim-3, belong to a distinctive serine/threonine protein-kinase family. They are involved in cytokine-induced signal transduction and the development of lymphoid malignancies. Their kinase domains are highly homologous to one another, but share low sequence identity to other kinases. Specifically, there are two proline residues in the conserved hinge-region sequence ERPXPX separated by a residue that is non-conserved among Pim kinases. Full-length human Pim-1 kinase (1–313) was cloned and expressed in Escherichia coli as a GST-fusion protein and truncated to Pim-1 (14–313) by thrombin digestion during purification. The Pim-1 (14–313) protein was purified to high homogeneity and monodispersity. This protein preparation yielded small crystals in the initial screening and large crystals after optimization. The large crystals of apo Pim-1 enzyme diffracted to 2.1 Å resolution and belong to space group P65, with unit-cell parameters a = b = 95.9, c = 80.0 Å, β = 120° and one molecule per asymmetric unit.
doi:10.1107/S1744309104029963
PMCID: PMC1952393  PMID: 16508102
Pim-1 kinase
17.  Purification, crystallization and preliminary X-ray diffraction analysis of the Kelch-like motif region of mouse Keap1 
Keap1-DC (Kelch/double-glycine repeat and C-terminal region) of mouse Keap1 has been overexpressed in E. coli, purified and crystallized using the vapour-diffusion method.
Keap1 (Kelch-like ECH-associating protein 1) is a negative regulator of the Nrf2 transcription factor in the cytoplasm. The Kelch/DGR (double-glycine repeat) domain of Keap1 associates with Nrf2 as well as with actin filaments. A recombinant protein containing both the Kelch/DGR domain and the C-­terminal region of mouse Keap1 was expressed in Escherichia coli, purified to near-homogeneity and crystallized by the sitting-drop vapour-diffusion method. The crystal belongs to space group P61 or P65, with unit-cell parameters a = b = 102.95, c = 55.21 Å, and contains one molecule in the asymmetric unit. A complete diffraction data was collected to 2.25 Å resolution using an R-AXIS IV++ imaging plate mounted on an RA-Micro7 Cu Kα rotating-anode X-ray generator.
doi:10.1107/S1744309104032506
PMCID: PMC1952392  PMID: 16508120
Nrf2; transcription factor; Keap1; actin binding; antioxidants
18.  Crystallization of leucyl-tRNA synthetase complexed with tRNALeu from the archaeon Pyrococcus horikoshii  
The leucyl-tRNA synthetase (LeuRS) from P. horikoshii has been overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNALeu isoacceptors have been attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNALeu isoacceptor with the anticodon CAA was used.
All five tRNALeu isoacceptors from the archaeon Pyrococcus horikoshii have been transcribed in vitro and purified. The leucyl-tRNA synthetase (LeuRS) from P. horikoshii was overexpressed in Escherichia coli and purified, and cocrystallizations with each of the tRNALeu isoacceptors were attempted. Cocrystals were obtained by the hanging-drop vapour-diffusion method, but only when the tRNALeu isoacceptor with the anticodon CAA was used. Electrophoretic analyses revealed that the crystals contain both LeuRS and tRNALeu, suggesting that they are LeuRS–tRNALeu complex crystals. A data set diffracting to 3.3 Å resolution was collected from a single crystal at 100 K. The crystal belongs to the orthorhombic space group P21212, with unit-cell parameters a = 118.18, b = 120.55, c = 231.13 Å. The asymmetric unit is expected to contain two complexes of LeuRS–tRNALeu, with a corresponding crystal volume per protein weight of 2.9 Å3 Da−1 and a solvent content of 57.3%.
doi:10.1107/S1744309104021827
PMCID: PMC1952391  PMID: 16508082
LeuRS; tRNA; leucine; identity; Pyrococcus horikoshii; long variable loop
19.  Crystallization and preliminary X-ray analysis of the GST-fused human Bri3 N-terminal domain 
The crystallization of the polyproline-rich polypeptide from human Bri3 overexpressed as a GST-fusion protein in Escherichia coli is presented.
Bri3 is a recently identified proline-rich transmembrane polypeptide up-regulated during TNF-mediated inflammation and immunity. The polyproline-rich N-terminal (residues 1–60) domain of Bri3 was affinity-purified to homogeneity as a glutathione-S-transferase (GST) fusion protein. Crystals were obtained in ∼3 d by the equilibrium vapour-diffusion method from a solution containing 1.5–2.2 M ammonium sulfate and 0.1 M bis-tris pH 6.0. The crystals belong to space group P43212, with unit-cell parameters a = b = 91.66, c = 57.53 Å. An X-ray data set was collected to 1.6 Å resolution using synchrotron radiation, with an R sym of 0.058 and a completeness of 95.3%. There is one molecule of the fusion protein in the asymmetric unit, which corresponds to ∼35% solvent content.
doi:10.1107/S1744309104026739
PMCID: PMC1952390  PMID: 16508092
Bri3; transmembrane proteins
20.  Crystallization and preliminary X-ray diffraction analysis of human growth and differentiation factor 5 (GDF-5) 
Crystals of human growth and differentiation factor 5 are trigonal, belonging to space group P3121 or its enantiomer, with one molecule per asymmetric unit and diffract to 2.2 Å resolution.
Growth and differentiation factor 5 (GDF-5) belongs to the large TGF-β superfamily of secreted signalling proteins and plays a pivotal role in skeletal development during embryogenesis. The gene for human GDF-5 was cloned, expressed in Escherichia coli and purified to homogeneity. Crystals were obtained that diffracted to 2.2 Å resolution. A native data set was acquired, showing that the crystals belong to a trigonal space group, i.e. P3121 or P3221, with unit-cell parameters a = b = 97.1, c = 48.3 Å. Initial analysis suggest the presence of only one monomer in the asymmetric unit, resulting in a high solvent content of 72% in the crystal.
doi:10.1107/S1744309104031963
PMCID: PMC1952389  PMID: 16508114
human growth and differentiation factor 5; signalling proteins
21.  Crystallization and preliminary X-ray diffraction studies of the glutaminyl cyclase from Carica papaya latex 
The glutaminyl cyclase isolated from C. papaya latex has been crystallized using the hanging-drop method. Diffraction data have been collected at ESRF beamline BM14 and processed to 1.7 Å resolution.
In living systems, the intramolecular cyclization of N-terminal glutamine residues is accomplished by glutaminyl cyclase enzymes (EC 2.3.2.5). While in mammals these enzymes are involved in the synthesis of hormonal and neurotransmitter peptides, the physiological role played by the corresponding plant enzymes still remains to be unravelled. Papaya glutaminyl cyclase (PQC), a 33 kDa enzyme found in the latex of the tropical tree Carica papaya, displays an exceptional resistance to chemical and thermal denaturation as well as to proteolysis. In order to elucidate its enzymatic mechanism and to gain insights into the structural determinants underlying its remarkable stability, PQC was isolated from papaya latex, purified and crystallized by the hanging-drop vapour-diffusion method. The crystals belong to the orthorhombic space group P212121, with unit-cell parameters a = 62.82, b = 81.23, c = 108.17 Å and two molecules per asymmetric unit. Diffraction data have been collected at ESRF beamline BM14 and processed to a resolution of 1.7 Å.
doi:10.1107/S1744309104025904
PMCID: PMC1952388  PMID: 16508091
glutaminyl cyclase
22.  Expression, purification and preliminary crystallographic analysis of sucrose phosphate synthase (SPS) from Halothermothrix orenii  
The first crystallographic study of a sucrose phosphate synthase from H. orenii, an organism that is both thermophilic and halophilic, is reported. The protein crystal diffracts X-rays to 3.01 Å.
This is the first report of the crystallization of a sucrose phosphate synthase (SPS; EC 2.4.1.14). It also constitutes the first study of a sucrose phosphate synthase from a non-photosynthetic thermohalophilic anaerobic bacterium, Halothermothrix orenii. The purified recombinant spsA protein has been crystallized in the monoclinic space group C2, with unit-cell parameters a = 154.2, b = 47.9, c = 72.3 Å, β = 103.16°, using the hanging-drop vapour-diffusion method. The crystal diffracts X-rays to a resolution limit of 3.01 Å. Heavy-metal and halide-soaking trials are currently in progress to solve the structure.
doi:10.1107/S174430910403091X
PMCID: PMC1952387  PMID: 16508108
sucrose phosphate synthase; Halothermothrix orenii; thermophiles; halophiles; sucrose metabolism
23.  Structure of a putative trans-editing enzyme for prolyl-tRNA synthetase from Aeropyrum pernix K1 at 1.7 Å resolution 
The three-dimensional structure of the APE2540 protein from A. pernix K1 has been determined by the multiple anomalous dispersion method at 1.7 Å resolution. The structure includes two monomers in the asymmetric unit and shares structural similarity with the YbaK protein or cysteinyl-tRNAPro deacylase from H. influenzae.
The crystal structure of APE2540, the putative trans-editing enzyme ProX from Aeropyrum pernix K1, was determined in a high-throughput manner. The crystal belongs to the monoclinic space group P21, with unit-cell parameters a = 47.4, b = 58.9, c = 53.6 Å, β = 106.8°. The structure was solved by the multiwavelength anomalous dispersion method at 1.7 Å and refined to an R factor of 16.8% (R free = 20.5%). The crystal structure includes two protein molecules in the asymmetric unit. Each monomer consists of eight β-strands and seven α-helices. A structure-homology search revealed similarity between the trans-editing enzyme YbaK (or cysteinyl-tRNAPro deacylase) from Haemophilus influenzae (HI1434; 22% sequence identity) and putative ProX proteins from Caulobacter crescentus (16%) and Agrobacterium tumefaciens (21%).
doi:10.1107/S1744309104032555
PMCID: PMC1952386  PMID: 16508081
trans-editing enzymes; APE2540
24.  Sequence-induced trimerization of phospholipase A2: structure of a trimeric isoform of PLA2 from common krait (Bungarus caeruleus) at 2.5 Å resolution 
The structure of a novel trimeric isoform of phospholipase A2 has been determined at 2.5 Å resolution. The trimer formation occurs in such a way that the active sites of all the three molecules are fully exposed to the solvent, making the trimer a highly potent enzymatic unit.
The venom of the common Indian krait (Bungarus caeruleus) contains about a dozen isoforms of phospholipase A2 (PLA2), which exist in different oligomeric forms as well as in complexes with low-molecular-weight ligands. The basic objective of multimerization and complexation is either to inactivate PLA2 in the venom for long-term storage, to generate a new PLA2 function or to make a more lethal assembly. The current isoform was isolated from the venom of B. caeruleus. Dynamic light-scattering studies indicated the presence of a stable trimeric association of this PLA2. Its primary sequence was determined by cDNA cloning. The purified protein was crystallized with 2.8 M NaCl as a precipitating agent using the sitting-drop vapour-diffusion method. The crystals belonged to the monoclinic space group C2, with unit-cell parameters a = 80.9, b = 80.5, c = 57.1 Å, β = 90.3°. The structure was refined to a final R factor of 0.198. This is a novel trimeric PLA2 structure in which the central pore formed by the association of three molecules is filled with water molecules. The interactions across the pore take place via multiple water bridges primarily to the side chains of Arg, Lys and Thr residues. Approximately 12% of the total solvent-accessible surface area is buried in the core of the trimer. The active sites of all three molecules are located on the surface and are fully exposed to the solvent, resulting in a highly potent enzymatic unit.
doi:10.1107/S1744309104025503
PMCID: PMC1952385  PMID: 16508078
phospholipase A2; oligomerization; aggregation
25.  Crystallization and preliminary crystallographic analysis of the nickel-responsive regulator NikR from Pyrococcus horikoshii  
Crystals of P. horikoshii NikR have been obtained in the presence and absence of Ni2+ and characterized by X-ray diffraction.
The nickel-responsive repressor from Pyrococcus horikoshii OT3 (PhNikR) has been crystallized in the apo form (PhNikR-apo) and two nickel-bound forms (PhNikR-Ni-1 and PhNikR-Ni-2). The PhNikR-apo crystals belong to space group P21, with unit-cell parameters a = 75.78, b = 54.32, c = 77.28 Å, β = 116.07°, and diffract to 2.2 Å. The PhNikR-Ni-1 crystals belong to space group P41212, with unit-cell parameters a = b = 99.89, c = 97.98 Å, and diffract to 3.0 Å and the PhNikR-Ni-2 crystals belong to space group P3121 or P3221, with unit-cell parameters a = b = 109.95, c = 79.0 Å, and diffract to 2.1 Å. The crystals obtained were suitable for detailed structural studies.
doi:10.1107/S1744309104025473
PMCID: PMC1952384  PMID: 16508086
NikR; nickel-responsive repressor

Results 1-25 (46)