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1.  Two Rhizobial Strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, Encode Haloalkane Dehalogenases with Novel Structures and Substrate Specificities 
Haloalkane dehalogenases are key enzymes for the degradation of halogenated aliphatic pollutants. Two rhizobial strains, Mesorhizobium loti MAFF303099 and Bradyrhizobium japonicum USDA110, have open reading frames (ORFs), mlr5434 and blr1087, respectively, that encode putative haloalkane dehalogenase homologues. The crude extracts of Escherichia coli strains expressing mlr5434 and blr1087 showed the ability to dehalogenate 18 halogenated compounds, indicating that these ORFs indeed encode haloalkane dehalogenases. Therefore, these ORFs were referred to as dmlA (dehalogenase from Mesorhizobium loti) and dbjA (dehalogenase from Bradyrhizobium japonicum), respectively. The principal component analysis of the substrate specificities of various haloalkane dehalogenases clearly showed that DbjA and DmlA constitute a novel substrate specificity class with extraordinarily high activity towards β-methylated compounds. Comparison of the circular dichroism spectra of DbjA and other dehalogenases strongly suggested that DbjA contains more α-helices than the other dehalogenases. The dehalogenase activity of resting cells and Northern blot analyses both revealed that the dmlA and dbjA genes were expressed under normal culture conditions in MAFF303099 and USDA110 strain cells, respectively.
doi:10.1128/AEM.71.8.4372-4379.2005
PMCID: PMC1183339  PMID: 16085827
2.  Crystals of DhaA mutants from Rhodococcus rhodochrous NCIMB 13064 diffracted to ultrahigh resolution: crystallization and preliminary diffraction analysis 
Three mutants of the haloalkane dehalogenase DhaA derived from R. rhodochrous NCIMB 13064 were crystallized and diffracted to ultrahigh resolution.
The enzyme DhaA from Rhodococcus rhodochrous NCIMB 13064 belongs to the haloalkane dehalogenases, which catalyze the hydrolysis of haloalkanes to the corresponding alcohols. The haloalkane dehalogenase DhaA and its variants can be used to detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Three mutants named DhaA04, DhaA14 and DhaA15 were constructed in order to study the importance of tunnels connecting the buried active site with the surrounding solvent to the enzymatic activity. All protein mutants were crystallized using the sitting-drop vapour-diffusion method. The crystals of DhaA04 belonged to the orthorhombic space group P212121, while the crystals of the other two mutants DhaA14 and DhaA15 belonged to the triclinic space group P1. Native data sets were collected for the DhaA04, DhaA14 and DhaA15 mutants at beamline X11 of EMBL, DESY, Hamburg to the high resolutions of 1.30, 0.95 and 1.15 Å, respectively.
doi:10.1107/S1744309108002066
PMCID: PMC2374176  PMID: 18259069
ultrahigh resolution; haloalkane dehalogenase DhaA mutants; Rhodococcus rhodochrous
3.  Crystallization and preliminary X-ray analysis of the haloalkane dehalogenase DatA from Agrobacterium tumefaciens C58 
The haloalkane dehalogenase DatA from A. tumefaciens C58 was expressed, purified and crystallized by the sitting-drop vapour-diffusion method. X-ray diffraction data were collected to 1.70 Å resolution.
Haloalkane dehalogenases are enzymes that catalyze the hydrolytic reaction of a wide variety of haloalkyl substrates to form the corresponding alcohol and hydrogen halide products. DatA from Agrobacterium tumefaciens C58 is a haloalkane dehalogenase that has a unique pair of halide-binding residues, asparagine (Asn43) and tyrosine (Tyr109), instead of the asparagine and tryptophan that are conserved in other members of the subfamily. DatA was expressed in Escherichia coli, purified and crystallized using the sitting-drop vapour-diffusion method with a reservoir solution consisting of 0.1 M CHES pH 8.6, 1.0 M potassium sodium tartrate, 0.2 M lithium sulfate, 0.01 M barium chloride. X-ray diffraction data were collected to 1.70 Å resolution. The space group of the crystal was determined as the primitive tetragonal space group P422, with unit-cell parameters a = b = 123.7, c = 88.1 Å. The crystal contained two molecules in the asymmetric unit.
doi:10.1107/S1744309112013942
PMCID: PMC3370902  PMID: 22684062
haloalkane dehalogenases; bioremediation
4.  Crystallization and crystallographic analysis of the Rhodococcus rhodochrous NCIMB 13064 DhaA mutant DhaA31 and its complex with 1,2,3-trichloropropane 
A mutant of the haloalkane dehalogenase DhaA (DhaA31) from R. rhodochrous NCIMB 13064 and its complex with 1,2,3-trichloropropane were crystallized and the crystals diffracted to high resolution.
Haloalkane dehalogenases hydrolyze carbon–halogen bonds in a wide range of halogenated aliphatic compounds. The potential use of haloalkane dehalogenases in bioremediation applications has stimulated intensive investigation of these enzymes and their engineering. The mutant DhaA31 was constructed to degrade the anthropogenic compound 1,2,3-trichloropropane (TCP) using a new strategy. This strategy enhances activity towards TCP by decreasing the accessibility of the active site to water molecules, thereby promoting formation of the activated complex. The structure of DhaA31 will help in understanding the structure–function relationships involved in the improved dehalogenation of TCP. The mutant protein DhaA31 was crystallized by the sitting-drop vapour-diffusion technique and crystals of DhaA31 in complex with TCP were obtained using soaking experiments. Both crystals belonged to the triclinic space group P1. Diffraction data were collected to high resolution: to 1.31 Å for DhaA31 and to 1.26 Å for DhaA31 complexed with TCP.
doi:10.1107/S1744309111001254
PMCID: PMC3053171  PMID: 21393851
haloalkane dehalogenases; DhaA; Rhodococcus rhodochrous
5.  Crystallization and initial X-ray diffraction studies of the flavoenzyme NAD(P)H:(acceptor) oxidoreductase (FerB) from the soil bacterium Paracoccus denitrificans  
The flavin-dependent enzyme FerB from P. denitrificans has been purified and both native and SeMet-substituted FerB have been crystallized. The two variants crystallized in two different crystallographic forms belonging to the monoclinic space group P21 and the orthorhombic space group P21212, respectively. X-ray diffraction data were collected to 1.75 Å resolution for both forms.
The flavin-dependent enzyme FerB from Paracoccus denitrificans reduces a broad range of compounds, including ferric complexes, chromate and most notably quinones, at the expense of the reduced nicotinamide adenine dinucleotide cofactors NADH or NADPH. Recombinant unmodified and SeMet-substituted FerB were crystallized under similar conditions by the hanging-drop vapour-diffusion method with microseeding using PEG 4000 as the precipitant. FerB crystallized in several different crystal forms, some of which diffracted to approximately 1.8 Å resolution. The crystals of native FerB belonged to space group P21, with unit-cell parameters a = 61.6, b = 110.1, c = 65.2 Å, β = 118.2° and four protein molecules in the asymmetric unit, whilst the SeMet-substituted form crystallized in space group P21212, with unit-cell parameters a = 61.2, b = 89.2, c = 71.5 Å and two protein molecules in the asymmetric unit. Structure determination by the three-wavelength MAD/MRSAD method is now in progress.
doi:10.1107/S1744309110005099
PMCID: PMC2852337  PMID: 20383015
flavoenzymes; quinone reductases; Paracoccus denitrificans
6.  Crystallization and preliminary X-ray diffraction studies of the putative haloalkane dehalogenase DppA from Plesiocystis pacifica SIR-I 
The crystallization and preliminary X-ray diffraction studies of DppA from P. pacifica SIR-I are reported.
DppA from Plesiocystis pacifica SIR-I is a putative haloalkane dehalogenase (EC 3.8.1.5) and probably catalyzes the conversion of halogenated alkanes to the corresponding alcohols. The enzyme was expressed in Escherichia coli BL21 and purified to homogeneity by ammonium sulfate precipitation and reversed-phase and ion-exchange chromatography. The DppA protein was crystallized by the vapour-diffusion method and protein crystals suitable for data collection were obtained in the orthorhombic space group P21212. The DppA crystal diffracted X-rays to 1.9 Å resolution using an in-house X-ray generator.
doi:10.1107/S1744309110018932
PMCID: PMC2898472  PMID: 20606284
haloalkane dehalogenases; Plesiocystis pacifica SIR-I
7.  Crystallization and preliminary X-ray diffraction analysis of the wild-type haloalkane dehalogenase DhaA and its variant DhaA13 complexed with different ligands 
Crystals of the wild-type haloalkane dehalogenase DhaA derived from R. rhodochrous NCIMB 13064 and of its catalytically inactive variant DhaA13 were grown in the presence of various ligands and diffraction data were collected to high and atomic resolution.
Haloalkane dehalogenases make up an important class of hydrolytic enzymes which catalyse the cleavage of carbon–halogen bonds in halogenated aliphatic compounds. There is growing interest in these enzymes owing to their potential use in environmental and industrial applications. The haloalkane dehalogenase DhaA from Rhodococcus rhodochrous NCIMB 13064 can slowly detoxify the industrial pollutant 1,2,3-trichloropropane (TCP). Structural analysis of this enzyme complexed with target ligands was conducted in order to obtain detailed information about the structural limitations of its catalytic properties. In this study, the crystallization and preliminary X-ray analysis of complexes of wild-type DhaA with 2-propanol and with TCP and of complexes of the catalytically inactive variant DhaA13 with the dye coumarin and with TCP are described. The crystals of wild-type DhaA were plate-shaped and belonged to the triclinic space group P1, while the variant DhaA13 can form prism-shaped crystals belonging to the orthorhombic space group P212121 as well as plate-shaped crystals belonging to the triclinic space group P1. Diffraction data for crystals of wild-type DhaA grown from crystallization solutions with different concentrations of 2-propanol were collected to 1.70 and 1.26 Å resolution, respectively. A prism-shaped crystal of DhaA13 complexed with TCP and a plate-shaped crystal of the same variant complexed with the dye coumarin diffracted X-rays to 1.60 and 1.33 Å resolution, respectively. A crystal of wild-type DhaA and a plate-shaped crystal of DhaA13, both complexed with TCP, diffracted to atomic resolutions of 1.04 and 0.97 Å, respectively.
doi:10.1107/S1744309110051286
PMCID: PMC3034621  PMID: 21301099
haloalkane dehalogenases; DhaA; Rhodococcus rhodochrous; microseeding; atomic resolution
8.  Crystallization and preliminary crystallographic analysis of the catechol 2,3-dioxygenase PheB from Bacillus stearothermophilus BR219 
PheB, an extradiol-cleaving catecholic dioxygenase, was crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The crystal belongs to the orthorhombic system, space group P212121, and diffracts to 2.3 Å resolution.
Class II extradiol-cleaving catecholic dioxygenase, a key enzyme of aromatic compound degradation in bacteria, cleaves the aromatic ring of catechol by adding two O atoms. PheB is one of the class II extradiol-cleaving catecholic dioxygenases and shows a high substrate specificity for catechol derivatives, which have one aromatic ring. In order to reveal the mechanism of the substrate specificity of PheB, PheB has been crystallized by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The space group of the obtained crystal was P212121, with unit-cell parameters a = 65.5, b = 119.2, c = 158.7 Å. The crystal diffracted to 2.3 Å resolution.
doi:10.1107/S174430910504323X
PMCID: PMC2150948  PMID: 16511281
extradiol-type dioxygenase; non-haem iron; thermostable proteins
9.  Crystallization and preliminary crystallographic study of a trypsin-resistant catalytic domain of human calcineurin 
A trypsin-resistant catalytic domain of human calcineurin α (A subunit, residues 20–347) was crystallized in space group P21212. An X-ray diffraction data set was collected to 2.87 Å resolution and the structure was solved by molecular replacement.
Calcineurin, a Ca2+/calmodulin-dependent serine/threonine protein phosphatase, plays a key role in a number of cellular pathways, including T-cell activation, and is an important molecular target of the immunosuppressive drugs cyclosporin A and FK506. To understand the structural basis underlying the activation of calcineurin by calmodulin, X-ray crystallography was employed to solve the three-dimensional structure of the free calcineurin catalytic domain (residues 20–347 of the A subunit). To accomplish this, a bacterially expressed glutathione S-­transferase (GST) fusion protein of the human calcineurin catalytic domain was first purified by GST-affinity chromatography. After limited digestion by trypsin, the catalytic domain (Cncat) was purified using anion-exchange and size-exclusion chromatography. Crystallization of Cncat was achieved by the hanging-drop vapour-diffusion method at pH 6.5 using PEG 6000 as precipitant. The diffraction results showed that the Cncat crystal belonged to the orthorhombic space group P21212, with unit-cell parameters a = 161.6, b = 87.4, c = 112.0 Å. There are four Cncat molecules in the asymmetric unit, with 49.5% solvent content. An X-ray diffraction data set was collected to 2.87 Å resolution and a clear molecular-replacement solution was obtained. The active site of Cncat is open to the solvent channels in the crystal packing.
doi:10.1107/S1744309112007890
PMCID: PMC3374516  PMID: 22691791
human calcineurin; catalytic domain
10.  Expression, purification and preliminary X-ray characterization of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1 
A recombinant form of dl-2-haloacid dehalogenase from Methylobacterium sp. CPA1 has been expressed in E. coli, purified and crystallized. The crystal belongs to space group P63. Diffraction data have been collected to 1.75 Å resolution.
dl-2-Haloacid dehalogenase from Methylobacterium sp. CPA1 (dl-DEX Mb) is a unique enzyme that catalyzes the dehalogenation reaction without the formation of an ester intermediate. A recombinant form of dl-DEX Mb has been expressed in Escherichia coli, purified and crystallized using the hanging-drop vapour-diffusion method. The crystal belongs to the hexagonal space group P63, with unit-cell parameters a = b = 186.2, c = 114.4 Å. The crystals are likely to contain between four and eight monomers in the asymmetric unit, with a V M value of 4.20–2.10 Å3 Da−1. A self-rotation function revealed peaks on the χ = 180° section. X-ray data have been collected to 1.75 Å resolution.
doi:10.1107/S1744309107027273
PMCID: PMC2335131  PMID: 17620717
dl-2-haloacid dehalogenase; dehalogenases
11.  Crystallization and preliminary X-ray diffraction analysis of the metalloregulatory protein DtxR from Thermoplasma acidophilum  
Orthorhombic crystals of DtxR from T. acidophilum have been obtained. X-ray data were collected to 1.8 Å resolution using synchrotron radiation.
The diphtheria toxin repressor (DtxR) is a metal-ion-dependent transcriptional regulator which regulates genes encoding proteins involved in metal-ion uptake to maintain metal-ion homeostasis. DtxR from Thermoplasma acidophilum was cloned and overexpressed in Escherichia coli. Crystals of N-terminally His-tagged DtxR were obtained by hanging-drop vapour diffusion and diffracted to 1.8 Å resolution. DtxR was crystallized at 296 K using polyethylene glycol 4000 as a precipitant. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 61.14, b = 84.61, c = 46.91 Å, α = β = γ = 90°. The asymmetric unit contained approximately one monomer of DtxR, giving a crystal volume per mass (V M) of 2.22 Å3 Da−1 and a solvent content of 44.6%.
doi:10.1107/S1744309111051700
PMCID: PMC3274395  PMID: 22297991
DtxR; transcriptional regulators; metalloregulatory proteins; Thermoplasma acidophilum
12.  Crystallization and preliminary X-ray crystallographic study of GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, in complex with translation elongation factor P 
GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, has been overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant.
GenX, a lysyl-tRNA synthetase paralogue from Escherichia coli, was overexpressed in E. coli, purified by three chromatographic steps and cocrystallized with a lysyl adenylate analogue (LysAMS) by the hanging-drop vapour-diffusion method using PEG 4000 as a precipitant. The GenX–LysAMS crystals belonged to the triclinic space group P1, with unit-cell parameters a = 54.80, b = 69.15, c = 94.08 Å, α = 95.47, β = 106.51, γ = 90.46°, and diffracted to 1.9 Å resolution. Furthermore, GenX was cocrystallized with translation elongation factor P (EF-­P), which is believed to be a putative substrate of GenX, and LysAMS using PEG 4000 and ammonium sulfate as precipitants. The GenX–EF-P–LysAMS crystals belonged to the monoclinic space group P21, with unit-cell parameters a = 105.93, b = 102.96, c = 119.94 Å, β = 99.4°, and diffracted to 2.5 Å resolution. Structure determination of the E. coli GenX–LysAMS and GenX–EF-P–LysAMS complexes by molecular replacement was successful and structure refinements are now in progress.
doi:10.1107/S1744309110032008
PMCID: PMC2935242  PMID: 20823541
GenX; lysyl-tRNA synthetase paralogue; translation elongation factor P
13.  Purification, crystallization and preliminary crystallographic analysis of DehIVa, a dehalogenase from Burkholderia cepacia MBA4 
The dhlIVa gene coding for DehIVa was expressed in Escherichia coli and the protein was purified and crystallized using the hanging-drop method.
DehIVa is one of two dehalogenases produced by the soil- and water-borne bacterium Burkholderia cepacia MBA4. It acts to break down short-chain halogenated aliphatic acids through a nucleophilic attack and subsequent hydrolysis of an enzyme–substrate intermediate to remove the halide ions from l-enantiomers substituted at the C2 position (e.g l-2-monochloropropionic acid). Dehalogenases are an important group of enzymes that are responsible for breaking down a diverse range of halogenated environmental pollutants. The dhlIVa gene coding for DehIVa was expressed in Escherichia coli and the protein was purified and crystallized using the hanging-drop method. Crystals grown in PEG 4000 and ammonium sulfate diffracted to 3.1 Å. The crystals had a primitive hexagonal unit cell, with unit-cell parameters a = b = 104.2, c = 135.8 Å, α = β = 90, γ = 120°. Determining this structure will provide valuable insights into the characterization of the catalytic mechanisms of this group of enzymes.
doi:10.1107/S1744309105002472
PMCID: PMC1952274  PMID: 16511015
dehalogenases; Burkholderia cepacia MBA4; DehIVa; halogenated organic acids
14.  Purification, crystallization and preliminary X-ray diffraction analysis of human enolase-phosphatase E1 
Recombinant human E1 enzyme has been crystallized using the hanging-drop vapour-diffusion method and diffraction-quality crystals were grown at 291 K using PEG 4000 as precipitant.
Enolase-phosphatase E1 (MASA) is a bifunctional enzyme in the ubiquitous methionine-salvage pathway and catalyzes the continuous reaction of 2,3-­diketo-5-methylthio-1-phosphopentane to yield the acireductone metabolite. Recombinant human E1 enzyme has been crystallized using the hanging-drop vapour-diffusion method and diffraction-quality crystals were grown at 291 K using PEG 4000 as precipitant. Diffraction data were collected to 1.7 Å resolution from SeMet-derivative crystals at 100 K using synchrotron radiation. The crystals belong to space group P212121, with unit-cell parameters a = 54.02, b = 57.55, c = 87.32 Å. The structure was subsequently solved by the multi-wavelength anomalous diffraction (MAD) phasing method.
doi:10.1107/S174430910501184X
PMCID: PMC1952300  PMID: 16511085
enolase-phosphatase E1; methionine salvage
15.  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
16.  Crystallization and preliminary X-ray diffraction studies of choline-binding protein F from Streptococcus pneumoniae  
The modular choline-binding protein F (CbpF) from S. pneumoniae has been crystallized by the hanging-drop vapour-diffusion method. A SAD data set from a gadolinium-complex derivative has been collected to 2.1 Å resolution.
Choline-binding protein F (CbpF) is a modular protein that is bound to the pneumococcal cell wall through noncovalent interactions with choline moieties of the bacterial teichoic and lipoteichoic acids. Despite being one of the more abundant proteins on the surface, along with the murein hydrolases LytA, LytB, LytC and Pce, its function is still unknown. CbpF has been crystallized using the hanging-drop vapour-diffusion method at 291 K. Diffraction-quality orthorhombic crystals belong to space group P21212, with unit-cell parameters a = 49.13, b = 114.94, c = 75.69 Å. A SAD data set from a Gd-HPDO3A-derivatized CbpF crystal was collected to 2.1 Å resolution at the gadolinium L III absorption edge using synchrotron radiation.
doi:10.1107/S1744309107035865
PMCID: PMC2376322  PMID: 17768343
choline-binding protein F; Streptococcus pneumoniae
17.  Crystallization and preliminary X-ray analysis of a glucansucrase from the dental caries pathogen Streptococcus mutans  
In this study, the glucansucrase from the dental caries pathogen S. mutans was purified and crystallized by the hanging-drop vapour-diffusion method using ammonium sulfate as a precipitant.
Glucansucrases encoded by Streptococcus mutans play essential roles in the synthesis of sticky dental plaques. Based on amino-acid sequence similarity, glucansucrases are classified as members of glycoside hydrolase family 70 (GH 70). Data on the crystal structure of GH 70 glucansucrases have yet to be reported. Here, the GH 70 glucansucrase GTF-SI from S. mutans was overexpressed in Escherichia coli strain BL21 (DE3), purified to homogeneity and crystallized using the hanging-drop vapour-diffusion method. Orthorhombic GTF-SI crystals belonging to space group P21212 were obtained. A diffraction data set was collected to 2.1 Å resolution.
doi:10.1107/S1744309110029714
PMCID: PMC2935234  PMID: 20823533
glucansucrase; dental caries; Streptococcus mutans
18.  Heterologous expression, purification, crystallization and preliminary X-ray analysis of raucaffricine glucosidase, a plant enzyme specifically involved in Rauvolfia alkaloid biosynthesis 
Raucaffricine glucosidase, an enzyme involved in the biosynthesis of monoterpenoid indole alkaloids in the plant Rauvolfia serpentina, was crystallized by the hanging-drop vapour-diffusion method using PEG4000 as precipitant. The crystals diffract to 2.3 Å resolution and belong to space group I222.
Raucaffricine glucosidase (RG) is an enzyme that is specifically involved in the biosynthesis of indole alkaloids from the plant Rauvolfia serpentina. After heterologous expression in Escherichia coli cells, crystals of RG were obtained by the hanging-drop vapour-diffusion technique at 293 K with 0.3 M ammonium sulfate, 0.1 M sodium acetate pH 4.6 buffer and 11% PEG 4000 as precipitant. Crystals belong to space group I222 and diffract to 2.30 Å, with unit-cell parameters a = 102.8, b = 127.3, c = 215.8 Å.
doi:10.1107/S174430910600457X
PMCID: PMC2197181  PMID: 16511316
raucaffricine glucosidase; indole alkaloid metabolism; biosynthesis; Rauvolfia serpentina
19.  Crystallization and preliminary X-ray analysis of 5-­keto-d-gluconate reductase from Gluconobacter suboxydans IFO12528 complexed with 5-keto-d-gluconate and NADPH 
NADPH-dependent 5-keto-d-gluconate reductase from G. suboxydans IFO12528 (5KGR) was expressed, purified and crystallized with 5-keto-d-gluconate and NADPH using the sitting-drop vapour-diffusion method. Crystals of the 5KGR–NADPH complex and of the 5KGR–NADPH–5-keto-d-gluconate complex diffracted X-rays to 1.75 and 2.26 Å resolution, respectively.
NADPH-dependent 5-keto-d-gluconate reductase from Gluconobacter suboxy­dans IFO12528 (5KGR) catalyzes oxidoreduction between 5-keto-d-gluconate and d-gluconate with high specificity. 5KGR was expressed in Escherichia coli, purified and crystallized with 5-keto-d-gluconate and NADPH using the sitting-drop vapour-diffusion method at 288 K. A crystal of the 5KGR–NADPH complex was obtained using reservoir solution containing PEG 4000 as a precipitant and diffracted X-rays to 1.75 Å resolution. The crystal of the complex belonged to space group P42212, with unit-cell parameters a = b = 128.6, c = 62.9 Å. A crystal of the 5KGR–NADPH–5-keto-d-gluconate complex was prepared by soaking the 5KGR–NADPH complex crystal in reservoir solution supplemented with 100 mM 5-keto-d-gluconate and 10 mM NADPH for 20 min and diffracted X-rays to 2.26 Å resolution. The crystal of the ternary complex belonged to space group P42212, with unit-cell parameters a = b = 128.7, c = 62.5 Å. Both crystals contained two molecules in the asymmetric unit.
doi:10.1107/S1744309110043617
PMCID: PMC2998383  PMID: 21139224
5-keto-d-gluconate reductase; Gluconobacter suboxydans IFO12528; SDR family
20.  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
21.  Crystallization and preliminary X-ray diffraction analysis of hemextin A: a unique anticoagulant protein from Hemachatus haemachatus venom 
Crystals of hemextin A, a three-finger toxin isolated and purified from African Ringhals cobra (H. haemachatus), are orthorhombic, space group P212121, with unit-cell parameters a = 49.27, b = 49.51, c = 57.87 Å, and diffract to 1.5 Å resolution.
Hemextin A was isolated and purified from African Ringhals cobra (Hemachatus haemachatus). It is a three-finger toxin that specifically inhibits blood coagulation factor VIIa and clot formation and that also interacts with hemextin B to form a unique anticoagulant complex. Hemextin A was crystallized by the hanging-drop vapour-diffusion method by equilibration against 0.2 M ammonium acetate, 0.1 M sodium acetate trihydrate pH 4.6 and 30% PEG 4000 as the precipitating agent. The crystals belong to space group P212121, with unit-cell parameters a = 49.27, b = 49.51, c = 57.87 Å and two molecules in the asymmetric unit. They diffracted to 1.5 Å resolution at beamline X25 at BNL.
doi:10.1107/S1744309107034239
PMCID: PMC2335159  PMID: 17671372
snake-venom toxins; three-finger toxins; hemextin; anticoagulants
22.  Molecular cloning, overexpression, purification, crystallization and preliminary X-ray diffraction studies of histidinol phosphate aminotransferase (HisC2) from Mycobacterium tuberculosis  
HisC2 from M. tuberculosis was cloned, overexpressed, purified and crystallized and the crystals were characterized.
HisC2 from Mycobacterium tuberculosis was overexpressed in M. smegmatis and purified to homogeneity using nickel–nitrilotriacetic acid metal-affinity and gel-filtration chromatography. Diffraction-quality crystals were grown using the hanging-drop vapour-diffusion technique from a condition consisting of 7 mg ml−1 HisC2 (in 20 mM Tris pH 8.8, 50 mM NaCl and 5% glycerol), 1 M succinic acid pH 7.0, 0.1 M HEPES pH 7.0 and 1%(w/v) polyethylene glycol monomethyl ether 2000. The crystals belonged to the orthorhombic space group P21212, with unit-cell parameters a = 255.98, b = 77.09, c = 117.97 Å. X-ray diffraction data were recorded to 2.45 Å resolution from a single crystal using the in-house X-ray facility.
doi:10.1107/S1744309111045386
PMCID: PMC3253829  PMID: 22232166
HisC2; Mycobacterium tuberculosis; Mycobacterium smegmatis
23.  Purification, crystallization and preliminary X-ray characterization of a haemagglutinin from the seeds of Jatropha curcas  
A novel haemagglutinin from Jatropha curcas seeds is purified and crystallized. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121 and the crystals diffracted to 2.8 Å resolution at 103 K.
The plant Jatropha curcas (Euphorbiaceae) is an important source of biofuel from the inedible oil present in its toxic seeds. The toxicity arises from the presence of curcin, a ribosome-inactivating protein showing haemagglutination activity. In this communication, the purification, crystallization and preliminary X-ray characterization are reported of a small protein isolated from J. curcas seeds with a molecular mass of ∼10 kDa that agglutinates rabbit erythrocytes. The protein was crystallized using the hanging-drop vapour-diffusion method and also by the microbatch method in 72-well HLA plates, using PEG 8000 as the precipitant in both conditions. X-ray diffraction data collected from the rod-shaped crystals were processed in the orthorhombic space group P212121. The crystals diffracted to 2.8 Å resolution at 103 K.
doi:10.1107/S1744309111038218
PMCID: PMC3232132  PMID: 22139159
Jatropha curcas; haemagglutinins
24.  Expression, purification, crystallization and preliminary X-ray analysis of perakine reductase, a new member of the aldo-keto reductase enzyme superfamily from higher plants 
Perakine reductase, a novel member of the aldo-keto reductase enzyme superfamily of higher plants, is involved in the biosynthesis of monoterpenoid indole alkaloids in the Indian medicinal plant Rauvolfia serpentina. The enzyme has been crystallized in C-centered orthorhombic space group and diffracts to 2.0 Å resolution.
Perakine reductase (PR) is a novel member of the aldo-keto reductase enzyme superfamily from higher plants. PR from the plant Rauvolfia serpentina is involved in the biosynthesis of monoterpenoid indole alkaloids by performing NADPH-dependent reduction of perakine, yielding raucaffrinoline. However, PR can also reduce cinnamic aldehyde and some of its derivatives. After heterologous expression of a triple mutant of PR in Escherichia coli, crystals of the purified and methylated enzyme were obtained by the hanging-drop vapour-diffusion technique at 293 K with 100 mM sodium citrate pH 5.6 and 27% PEG 4000 as precipitant. Crystals belong to space group C2221 and diffract to 2.0 Å, with unit-cell parameters a = 58.9, b = 93.0, c = 143.4 Å.
doi:10.1107/S174430910605041X
PMCID: PMC2225361  PMID: 17142919
perakine reductase; indole alkaloid metabolism; biosynthesis; Rauvolfia serpentina
25.  Crystallization and preliminary crystallographic study of the yeast Malassezia sympodialis allergen Mala s 1 
Crystals of the M. sympodialis allergen Mala s 1 have been obtained using the hanging-drop vapour-diffusion method. A diffraction data set has been collected from native crystals to 1.35 Å resolution.
The opportunistic yeast Malassezia sympodialis can act as an allergen and elicit specific IgE- and T-cell reactivity in patients with atopic eczema. The first identified major allergen from M. sympodialis, Mala s 1, is present on the cell surface of the yeast. Recombinant Mala s 1 was expressed in Escherichia coli, purified and refolded in a soluble form. Crystals of Mala s 1 were obtained in 25% PEG 8K, 0.2 M (NH4)2SO4. Crystals belong to space group P21212, with unit-cell parameters a = 44.4, b = 163.7, c = 50.6 Å, and diffract to 1.35 Å resolution.
doi:10.1107/S1744309105041011
PMCID: PMC2150955  PMID: 16511273
Mala s 1; Malassezia sympodialis; recombinant allergen; atopic eczema

Results 1-25 (125942)