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In bacteria and eukaryotes, the last two steps of de novo purine biosynthesis are catalyzed by bifunctional purine-biosynthesis protein (PurH), which is composed of two functionally independent domains linked by a flexible region. The N-terminal domain possesses IMP cyclohydrolase activity and the C-terminal domain possesses aminoimidazole-4-carboxamide ribonucleotide transformylase activity. This study reports the expression, purification, crystallization and preliminary X-ray crystallographic analysis of PurH from Escherichia coli with an N-terminal His6 tag. The crystals diffracted to a maximum resolution of 3.05 Å and belonged to the monoclinic space group P21, with unit-cell parameters a = 76.37, b = 132.15, c = 82.64 Å, β = 111.86°. [ABSTRACT FROM AUTHOR]

Snake-venom thrombin-like enzymes (SVTLEs) are serine proteases that are widely distributed in snakes from the Crotalinae subfamily of the Viperidae. In contrast to other snake-venom serine proteases, they have a biochemical activity similar to that of thrombin and play an important role in the process of blood coagulation. However, SVTLEs cannot activate factor VIII, which is essential in blood-clot stabilization. Consequently, blood clots produced by SVTLEs are not stable and are cleared rapidly. This characteristic makes SVTLEs attractive as potential candidates for antithrombotic therapy. Saxthrombin, an SVTLE from Gloydius saxatilis, was purified and crystallized to obtain a high-quality crystal, from which data were acquired to 1.43 Å resolution. Preliminary X-ray diffraction analysis showed that the crystal belonged to space group C2, with unit-cell parameters a = 94.2, b = 52.2, c = 50.1 Å, β = 96.7°. The crystal structure was determined by molecular replacement and the final R factor was 18.69%; the Rfree was 20.01%. This is the first report of a crystal structure of an SVTLE. Saxthrombin belongs to the typical α/β-hydrolase fold of serine proteases. Its structure was compared with those of thrombin and other snake-venom serine proteases. The observed differences in the amino-acid composition of the loops surrounding the active site appear to contribute to different surface-charge distributions and thus alter the shape of the active-site cleft, which may explain the differences in substrate affinity. [ABSTRACT FROM AUTHOR]

Fungal laccases are oxidoreductases that belong to the multinuclear copper-containing oxidases. They are able to oxidize a wide range of substrates, preferably phenolic compounds, which makes them suitable for employment in the bioremediation of soil and water as well as in other biotechnological applications. Here, the structural analysis of natural laccase B (LacB) from Trametes sp. AH28-2 is presented. This structure provides the opportunity to study the natural post-translational modifications of the enzyme. The overall fold shows a high homology to those of previously analyzed laccases with known three-dimensional structure. However, LacB contains a new structural element, a protruding loop near the substrate-binding site, compared with the previously reported laccase structures. This unique structural feature may be involved in modulation of the substrate recognition of LacB. [ABSTRACT FROM AUTHOR]

Adenylate kinase plays a very important role in regulating adenylate species in the cell. Methanocaldococcus jannaschii is a rich resource of unique enzymes. Here, MJ0458, an adenylate kinase from M. jannaschii, was crystallized. A set of X-ray diffraction data to 2.70 Å resolution was collected on beamline BL-17U of the Shanghai Synchrotron Radiation Facility (SSRF). The crystal belonged to space group P41212 or P43212. The unit-cell parameters were a = b = 76.18, c = 238.70 Å, α = β = γ = 90°. [ABSTRACT FROM AUTHOR]

Synaptotagmin acts as the Ca2+ sensor for neural and endocrine exocytosis. Synaptotagmin 5 has been demonstrated to play a key role in the acquisition of cathepsin D and the vesicular proton ATPase and in Ca2+-dependent insulin exocytosis. The C2 domains modulate the interaction of synaptotagmin with the phospholipid bilayer of the presynaptic terminus and effector proteins such as the SNARE complex. This study reports the cloning, expression in Escherichia coli, purification, crystallization and preliminary X-ray analysis of the C2A domain of human synaptotagmin 5 with an N-terminal His6 tag. The crystals diffracted to 1.90 Å resolution and belonged to the hexagonal space group P65, with unit-cell parameters a = b = 93.97, c = 28.05 Å. A preliminary model of the protein structure has been built and refinement of the model is ongoing. [ABSTRACT FROM AUTHOR]

In Saccharomyces cerevisiae, TRM6 and TRM61 compose a tRNA methyltransferase which catalyzes the methylation of the N1 of adenine at position 58 in tRNAs, especially initiator methionine tRNA. TRM61 is the subunit that binds S-adenosyl- l-methionine and both subunits contribute to target tRNA binding. In order to elucidate the catalytic mechanism of TRM6-TRM61 and the mode of interaction between the two subunits, expression, purification, crystallization and X-ray diffraction analysis of the TRM6-TRM61 complex were performed in this study. The crystals diffracted to 2.80 Å resolution and belonged to the trigonal space group P3121 or P3221, with unit-cell parameters a = b = 139.14, c = 101.62 Å. [ABSTRACT FROM AUTHOR]

Nitrogen fixation is catalyzed by the nitrogenase complex in Azotobacter, which is composed of dinitrogenase and dinitrogenase reductase. Dinitrogenase is an α2β2 heterotetramer of the proteins NifD and NifK. Dinitrogenase reductase is a homodimer of the protein NifH. The expression of NifD/K and NifH nitrogenase homologues (named NflD/K and NflH for Nif-like D and H, respectively) has been detected in the non-nitrogen-fixing hyperthermophilic methanogen Methanocaldococcus jannaschii. Solving the structure of MjNifH1 may help in better understanding its function and may supply some clues to understanding the evolution of nitrogenase. The full-length protein with an additional His6 tag at the C-terminus was expressed, purified and crystallized by the hanging-drop vapour-diffusion method at 287 K. An X-ray diffraction data set was collected to a resolution of 3.3 Å. The crystal belonged to space group P4132, with unit-cell parameters a = b = c = 139.45 Å, and was estimated to contain one protein molecule per asymmetric unit. [ABSTRACT FROM AUTHOR]

Nitrogenases are protein complexes that are only found in Azotobacter and are required for biological nitrogen fixation. They are made up of a nitrogenase, which is a NifD2/NifK2 heterotetramer, and a nitrogenase reductase, which is a homodimer of NifH. Many homologues of nitrogenase have been found in various non-nitrogen-fixing prokaryotes; in particular, they are found in all known methanogens. This indicates that these homologues may play a role in methane production. Here, the cloning of NifH2, a homologue of the NifH nitrogenase component, from Methanocaldococcus jannaschii ( MjNifH2) and its expression in Escherichia coli with a polyhistidine tag, purification and crystallization are described. MjNifH2 crystals were obtained by the hanging-drop vapour-diffusion method and diffracted to a resolution limit of 2.85 Å. The crystals belonged to space group P2, with unit-cell parameters a = 64.01, b = 94.38, c = 98.08 Å, α = γ = 90, β = 98.85°. [ABSTRACT FROM AUTHOR]

5-Aminolaevulinic acid dehydratase (ALAD), a crucial enzyme in the biosynthesis of tetrapyrrole, catalyses the condensation of two 5-aminolaevulinic acid (ALA) molecules to form porphobilinogen (PBG). The gene encoding ALAD was amplified from genomic DNA of Bacillus subtilis and the protein was overexpressed in Escherichia coli strain BL21 (DE3). The protein was purified and crystallized with an additional MGSSHHHHHHSSGLVPRGSH- tag at the N-terminus of the target protein. Diffraction-quality single crystals were obtained by the hanging-drop vapour-diffusion method. An X-ray diffraction data set was collected at a resolution of 2.7 Å. [ABSTRACT FROM AUTHOR]

Escherichia coli YeaD (ecYeaD) is suggested to be a member of the galactose mutarotase-like superfamily. Galactose mutarotase is an enzyme that converts α-galactose to β-galactose. The known structures of these galactose mutarotase-like proteins are similar to those of galactose mutarotases, with the catalytic residues being conserved, but there are some differences between them in the substrate-binding pocket. In order to reveal the specificity of ecYeaD, a three-dimensional structure is essential. Full-length ecYeaD with an additional 6×His tag at the C-terminus was crystallized by the hanging-drop vapour-diffusion method using polyethylene glycol 4000 as a precipitant at 283 K. An X-ray diffraction data set was collected to a resolution of 1.9 Å from a single flash-cooled crystal that belonged to space group P212121. [ABSTRACT FROM AUTHOR]

The histone chaperone SET encoded by the SET gene, which is also known as template-activating factor Iβ (TAF-Iβ), is a multifunctional molecule that is involved in many biological phenomena such as histone binding, nucleosome assembly, chromatin remodelling, replication, transcription and apoptosis. A truncated SET/TAF-IβΔN protein that lacked the first 22 residues of the N-terminus but contained the C-terminal acidic domain and an additional His6 tag at the C-terminus was overexpressed in Escherichia coli and crystallized by the hanging-drop vapour-diffusion method using sodium acetate as precipitant at 283 K. The crystals diffracted to 2.7 Å resolution and belonged to space group P43212. [ABSTRACT FROM AUTHOR]