Crystallization and preliminary X-ray analysis of L-2-haloacid dehalogenase from Xanthobacter autotrophicus GJ10.

Haloacid dehalogenases are enzymes that cleave carbon-chlorine or carbon-bromine bonds of 2-haloalkanoates. X-ray-quality crystals of L-2-haloacid dehalogenase from the 1,2-dichloroethane-degrading bacterium Xanthobacter autotrophicus GJ10 have been grown at room temperature from 20% PEG 8000, 200 mM sodium formate at pH 6.8-7.0, using macroseeding techniques. The crystals, which diffract in the X-ray beam up to 2.0 A resolution, belong to the spacegroup C2221. Cell parameters are a = 58.8 A, b = 93.1 A, c = 84.2 A. A native data set to 2.3 A has been collected, with a completeness of 97% and an Rsym of 6.0%.     

Crystallization and preliminary X-ray crystallographic analysis of arylesterase from Pseudomonas fluorescens

Large crystals of arylesterase from Pseudomonas fluorescens have been grown at room temperature using ammonium sulfate as a precipitant. They grow to dimensions of 0.7 × 0.7 × 0.6 mm³ within a month. The crystals belong to the trigonal space group P3₁ (or P3₂), with unit cell dimensions of a= 147.12 Å and c= 131.08 Å. The asymmetric unit seems to contain six molecules of dimeric aryles-terase, with corresponding crystal volume per protein mass (VM ) of 2.53 ų/Da and solvent fraction of 51.5% by volume. The crystals diffract to at least 2.2 Å Bragg spacing when exposed to X-rays from a rotating-anode source. X-ray data have been collected to 2.9 Å Bragg spacing from native crystals. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic studies of UDP-N-acetylenolpyruvylglucosamine reductase.

The overexpression and purification of the second enzyme in Escherichia coli peptidoglycan biosynthesis, UDP-N-acetylenolpyruvylglucosamine reductase (MurB), provided sufficient protein to undertake crystallization and X-ray crystallographic studies of the enzyme. MurB crystallizes in 14-20% PEG 8000, 100 mM sodium cacodylate, pH 8.0, and 200 mM calcium acetate in the presence of its substrate UDP-N-acetylglucosamine enolpyruvate. Crystals of MurB belong to the tetragonal space group P4(1)2(1)2 with a = b = 49.6 A, c = 263.2 A, and alpha = beta = gamma = 90 degrees at -160 degrees C and diffract to at least 2.5 A. Screening for heavy atom derivatives has yielded a single site that is reactive with both methylmercury nitrate and Thimerosal.     

Crystallization and preliminary X-ray crystallographic studies of nonhistone region of macroH2A.1

Histone macroH2A has a novel hybrid structure consisting of a large nonhistone region and a region that closely resembles a full-length histone H2A. One key to understanding macroH2A function is determining the structure and function of its nonhistone region. The nonhistone region of one of the two known macroH2A subtypes was expressed in Escherichia coli and purified using affinity and molecular sieve chromatography. Crystals of the protein suitable for structural studies were grown from polyethylene glycol solutions by vapor equilibration techniques. The crystals belong to the hexagonal space group P6₄ (or its enantiomorph P6₂) with unit cell parameters: a = b = 106.2 Å, c = 125.9 Å, α = β = 90°, and γ = 120°. There are four molecules in the asymmetric unit. Self-rotation function studies revealed three twofold noncrystallographic rotation axes related approximately by 222 symmetry. These crystals have 47% solvent content and diffract to 3.8 Å resolution. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of ImmE7 protein of colicin E7

The ImmE7 protein, which can bind specifically to the DNase colicin E7 and neutralize its bactericidal activity, has been purified and crystallized in two different crystal forms by vapor diffusion method. The orthorhombic crystals belong to space group I222 or I2₁2₁2₁ and have unit cell dimensions a = 75.1 Å, b = 50.5 Å, and c = 45.4 Å. The second form is monoclinic space group P2₁ with ceil dimensions a = 29.3 Å, b = 102.7 Å, c = 53.0 Å and β = 91.5°. The orthorhombic crystals diffract to 1.8 Å resolution, and are suitable for high-resolution X-ray analysis. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic studies on Type III antifreeze protein.

Type III antifreeze protein, more specifically the recombinant QAE-Sephadex-binding isoform, has been crystallized in 50-55% saturated ammonium sulfate, 0.1 M sodium acetate, pH 4.0-4.5. The resultant crystals belong to the orthorhombic space group P212121 with a = 32.60 A, b = 39.00 A, and c = 46.57 A and diffract to at least 1.7 A. A set of 1.7-A native data has been collected, with completeness 93.4% and Rsym of 0.069. Initial screening for heavy-atom derivatives has yielded a Pt-bound derivative.     

Multi-template homology-based structural model of L-2-haloacid dehalogenase (DehL) from Rhizobium sp. RC1

Dehalogenases are of high interest due to their potential applications in bioremediation and in synthesis of various industrial products. DehL is an L-2-haloacid dehalogenase (EC 3.8.1.2) that catalyses the cleavage of halide ion from L-2-halocarboxylic acid to produce D-2-hydroxycarboxylic acid. Although DehL utilises the same substrates as the other L-2-haloacid dehalogenases, its deduced amino acid sequence is substantially different (<25%) from those of the rest L-2-haloacid dehalogenases. To date, the 3D structure of DehL is not available. This limits the detailed understanding of the enzyme’s reaction mechanism. The present work predicted the first homology-based model of DehL and defined its active site. The monomeric unit of the DehL constitutes α/β structure that is organised into two distinct structural domains: main and subdomains. Despite the sequence disparity between the DehL and other L-2-haloacid dehalogenases, its structural model share similar fold as the experimentally solved L-DEX and DehlB structures. The findings of the present work will play a crucial role in elucidating the molecular details of the DehL functional mechanism.     

Crystallization and preliminary X-ray crystallographic studies of ketosteroid isomerase from Pseudomonas putida biotype B

The Δ⁵-3-ketosteroid isomerase from Pseudomonas putida biotype B has been crystallized. The crystals belong to the space group P2₁2₁2₁ with unit cell dimensions of a = 36.48 Å, b = 74.30 Å, c = 96.02 Å, and contain one homodimer per asymmetric unit. Native diffraction data to 2.19 Å resolution have been obtained from one crystal at room temperature indicating that the crystals are quite suitable for structure determination by multiple isomorphous replacement.     

Crystallization and preliminary X-ray crystallographic analysis of phosphoribulokinase from Rhodobacter sphaeroides.

A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (PRK), expressed in Escherichia coli and isolated by affinity chromatography, was crystallized by the sitting drop vapor diffusion technique using NH4H2PO4 (pH 5.6) as the precipitating agent. PRK crystallizes in the cubic space group P432, with unit cell parameters a = b = c = 129.55 A. Based on the assumption of one 32-kDa monomer per asymmetric unit, the Vm value is 2.83 A3/Da. The octameric molecular symmetry is consistent with two planar tetramers stacked in a nearly eclipsed arrangement. A native data set has been collected to 2.6 A resolution.     

Overexpression and feasible purification of thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL

The gene encoding thermostable L-2-halo acid dehalogenase ofPseudomonas sp. YL was isolated, and its over-expression system was constructed. Gene library was prepared fromSau3AI fragments of total DNA fromPs. sp. YL, pUC118 as a vector andEscherichia coli JM109 as a host. The recombinant cells resistant to bromoacetate, a germicide, were isolated and shown to produce L-2-halo acid dehalogenase. Subsequently, subcloning was carried out with pKK223-3 as a vector, and the length of DNA inserted was reduced to 1.1 kbp. One of the subclones showed very high activity, and the amount of the dehalogenase produced corresponded to about 30% of the soluble protein. From 5 g (wet weight) of cells, 105 mg of dehalogenase was efficiently purified by heat treatment and DEAE-Toyopearl chromatography. This overexpression system provides a large amount of the thermostable enzyme to enable us to study the properties, structure and application of the enzyme.Abbreviations IPTG isopropyl -D-thiogalactopyranoside - KPB potassium phosphate buffer - SDS sodium dodecyl sulfate - X-gal 5-bromo-4-chloro-3-indolyl--D-galactoside     

Crystallization and preliminary X-ray crystallographic properties of Hsc20, a J-motif co-chaperone protein from Escherichia coli.

Hsc20 is a 20-kDa auxiliary protein that functions with the molecular chaperone Hsc66 in Escherichia coli. Crystals of Hsc20 suitable for X-ray diffraction analysis were grown using the hanging drop vapor diffusion technique in polyethylene glycol 400 containing dioxane as an additive to slow growth. The crystals are monoclinic and belong to the space group C2 with unit cell dimensions a = 125.4 A, b = 71.9 A, c = 68.8 A, and beta = 97.0 degrees. The crystals diffract to a minimum d-spacing of approximately 2.5 A resolution, and a native data set was collected to 2.7 A. The results of a self-rotation function analysis revealed threefold symmetry, suggesting three molecules of Hsc20 in the asymmetric unit and, hence, 12 molecules in the unit cell; this corresponds to a Vm value of 2.6 A3/Da and a solvent content of approximately 53% in the crystals. Structure determination by isomorphous replacement is in progress.     

Crystallization and preliminary X-ray diffraction studies of the human major histocompatibility antigen HLA-B27.

The class I major histocompatibility (MHC) antigen HLA-B27 was purified by immunoaffinity chromatography from the homozygous human B lymphoblastoid cell line LG-2. Detergent-soluble HLA-B27 was cleaved with the protease papain to remove the hydrophobic transmembrane region and the cytoplasmic tail. Crystals of the resulting water-soluble extracellular fragments were obtained in hanging drops by the vapor-diffusion method. The crystals are triclinic, space group P1, with unit cell dimensions a = 45.9 A, b = 71.0 A, c = 83.7 A, alpha = 79.4 degrees, beta = 88.5 degrees, gamma = 89.9 degrees, and diffract beyond 2.5 A resolution.     

Crystallization and preliminary X-ray crystallographic analysis of chitinase from barley seeds

Chitinase from barley seeds has been crystallized at room temperature using polyethylene glycol as precipitant. The crystal is monoclinic, belonging to the space group P2₁, with unit cell parameters of a = 69.43 Å, b = 44.55 Å, c = 81.41 Å, and β = 111.95 Å. The asymmetric unit seems to contain two molecules of chitinase with a corresponding crystal volume per protein mass (V_M) of 2.25 ų/Da and a solvent content of 45% by volume. The crystal diffracts to at least 2.0 Å with X-rays from a rotating anode source and is very stable in the X-ray beam. X-ray data have been collected to better than 2.2 Å Bragg spacing from a native crystal. © 1993 Wiley-Liss, Inc.     

Crystallization and preliminary X-ray crystallographic analysis of squalene-hopene cyclase from Alicyclobacillus acidocaldarius.

The membrane-associated protein squalene-hopene cyclase from Alicyclobacillus acidocaldarius was overexposed in Escherichia coli and purified by ion exchange and gel permeation chromatography. Crystals of three interrelated forms were grown by vapor diffusion under identical conditions. The crystals diffract to about 2.3 A resolution, but they are unstable in the X-ray beam. An interpretable heavy-atom derivative was obtained.     

Crystallization and preliminary X-ray crystallographic analysis of phospholipid transfer protein from maize seedlings

Phospholipid transfer protein from maize seedlings has been crystallized using trisodium citrate as precipitant. The crystal belongs to the orthorhombic space group P2₁2₁2₁ with unit cell dimensions of a = 24.46 Å, b = 49.97 Å, and c = 69.99 Å. The presence of one molecule in the asymmetric unit gives a crystal volume per protein mass (V_m) of 2.36 Å ³/Da and a solvent content of 48% by volume. The X-ray diffraction pattern extends at least to 1.6 Å Bragg spacing when exposed to both CuK_α and synchrotron X-rays. A set of X-ray data to approximately 1.9 Å Bragg spacing has been collected from a native crystal. © 1994 Wiley-Liss, Inc.     

Crystallization and preliminary diffraction studies of thioesterase II from rat mammary gland

Thioesterase II from rat mammary gland has been crystallized in the presence of decanoic acid by the vapor diffusion method. The crystals belong to the orthorhombic space group P2₁2₁2₁, and have cell dimensions, a = 52.7 Å, b = 78.0 Å, and c = 133.6 Å. The asymmetric unit likely consists of two protein monomers based on predictions from its calculated Matthews coefficient. Crystals typically diffract to at least 2.5 Å resolution and are suitable for X-ray crystallographic analysis. © 1995 Wiley-Liss, Inc.     

Bacterial DL-2-haloacid dehalogenase from Pseudomonas sp. strain 113: gene cloning and structural comparison with D- and L-2-haloacid dehalogenases.

DL-2-Haloacid dehalogenase from Pseudomonas sp. strain 113 (DL-DEX) catalyzes the hydrolytic dehalogenation of both D- and L-2-haloalkanoic acids to produce the corresponding L- and D-2-hydroxyalkanoic acids, respectively, with inversion of the C2 configuration. DL-DEX is a unique enzyme: it acts on the chiral carbon of the substrate and uses both enantiomers as equivalent substrates. We have isolated and sequenced the gene encoding DL-DEX. The open reading frame consists of 921 bp corresponding to 307 amino acid residues. No sequence similarity between DL-DEX and L-2-haloacid dehalogenases was found. However, DL-DEX had significant sequence similarity with D-2-haloacid dehalogenase from Pseudomonas putida AJ1, which specifically acts on D-2-haloalkanoic acids: 23% of the total amino acid residues of DL-DEX are conserved. We mutated each of the 26 residues with charged and polar side chains, which are conserved between DL-DEX and D-2-haloacid dehalogenase. Thr65, Glu69, and Asp194 were found to be essential for dehalogenation of not only the D- but also the L-enantiomer of 2-haloalkanoic acids. Each of the mutant enzymes, whose activities were lower than that of the wild-type enzyme, acted on both enantiomers of 2-haloacids as equivalent substrates in the same manner as the wild-type enzyme. We also found that each enantiomer of 2-chloropropionate competitively inhibits the enzymatic dehalogenation of the other. These results suggest that DL-DEX has a single and common catalytic site for both enantiomers.     

Crystallization and preliminary crystallographic study of human lithostathine

Crystals of human lithostathine, a pancreatic glycoprotein which inhibits the growth and nucleation of calcium carbonate crystals, were grown using PEG 4000 as the precipitating agent. The crystals belong to the hexagonal space group P6₁ (or its enantiomorph P6₅) and diffract to 1.55 Å resolution. There is one molecule in the asymmetric unit and the crystals have 39% solvent. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic analysis of an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei

The thermostable amylopullulanase from Pyrococcus woesei was crystallized. Crystals, suitable for a crystallographic analysis up to a size of 0.6 mm in their longest dimension, have been obtained by the vapor diffusion method in a solution containing polyethyleneglycol 4000 (PEG 4000), isopropanol, and Tris/Cl^? buffer pH 7.5. Crystals grown under these conditions form hexagonal rods and diffract to a maximum resolution of 3 Å. The crystals belong to the trigonal lattice type with the spacegroup P3₁21 or P3₂21, respectively, have the cell dimensions a = b = 96.8 Å, c = 196.2 Å, α = β = 90°,γ = 120°. The crystals have a theoretical packing density of 2.7 ų/Da, assuming one molecule with a molecular weight of 88.8 kDa in the asymmetric unit. Furthermore the self-rotation analysis of the dataset revealed only crystallographic symmetries. The merged native data of two crystals resulted in a 88% complete dataset. © 1995 Wiley-Liss, Inc.     

Crystallization and preliminary crystallographic study of human CksHs1: A cell cycle regulatory protein

The cell cycle regulatory protein CksHs1 has been crystallized in a form suitable for X-ray studies. CksHsl crystals were grown in the presence of vanadate, a phos-phatase inhibitor, but were also obtained with phosphate or tungstate as a cofactor. They belong to the hexagonal space group P6₁22 with unit cell dimensions: a=b=94 Å, c=131.6 Å, and γ =120. The crystals grown in the presence of vanadate diffract X-rays to at least 2.8 Å. Molecular replacement results from the homologous human CksHs2 structure reveal that a dimer forms the crystal habit, giving the unusual V_m value of 4.4 ų/Da or a solvent content of 72%. © 1995 Wiley-Liss, Inc.