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Early on, crystallography was a domain of mineralogy and mathematics and dealt mostly with symmetry properties and imaginary crystal lattices. This changed when Wilhelm Conrad Röntgen discovered X-rays in 1895, and in 1912, Max von Laue and his associates discovered that X-ray irradiated salt crystals would produce diffraction patterns that could reveal the internal atomic periodicity of the crystals. In the same year, the father-and-son team, Henry and Lawrence Bragg successfully solved the first crystal structure of sodium chloride and the era of modern crystallography began. Protein crystallography (PX) started some 20 years later with the pioneering work of British crystallographers. In the past 50–60 years, the achievements of modern crystallography and particularly those in PX have been due to breakthroughs in theoretical and technical advancements such as phasing and direct methods; to more powerful X-ray sources such as synchrotron radiation; to more sensitive and efficient X-ray detectors; to ever faster computers and to improvements in software. The exponential development of PX has been accelerated by the invention and applications of recombinant DNA technology that can yield nearly any protein of interest in large amounts and with relative ease. Novel methods, informatics platforms and technologies for automation and high-throughput have allowed the development of large-scale, high-efficiency macromolecular crystallography efforts in the field of structural genomics. Very recently, the X-ray free-electron laser sources and its applications in PX have shown great potential for revolutionizing the whole field again in the near future. [ABSTRACT FROM AUTHOR]

ABSTRACT The protein Smu.1393c from Streptococcus mutans is annotated as a putative α/β hydrolase, but it has low sequence identity to the structure-known α/β hydrolases. Here we present the crystal structure of Smu.1393c at 2.0 Å resolution. Smu.1393c has a fully open alkaline substrate pocket, whose conformation is unique among other similar hydrolase structures. Three residues, Ser101, His251, and Glu125, were identified as the active center of Smu.1393c. By screening a series of artificial hydrolase substrates, we demonstrated Smu.1393c had low carboxylesterase activity towards short-chain carboxyl esters, which provided a clue for exploring the in vivo function of Smu.1393c. Proteins 2014; 82:695-700. © 2013 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Abstract: Glucosamine-6-phosphate (GlcN6P) N-acetyltransferase 1 (GNA1) is a key enzyme in the pathway toward biosynthesis of UDP-N-acetylglucosamine, an important donor substrate for N-linked glycosylation. GNA1 catalyzes the formation of N-acetylglucosamine-6-phosphate (GlcNAc6P) from acetyl-CoA (AcCoA) and the acceptor substrate GlcN6P. Here, we report crystal structures of human GNA1, including apo GNA1, the GNA1-GlcN6P complex and an E156A mutant. Our work showed that GlcN6P binds to GNA1 without the help of AcCoA binding. Structural analyses and mutagenesis studies have shed lights on the charge distribution in the GlcN6P binding pocket, and an important role for Glu156 in the substrate binding. Hence, these findings have broadened our knowledge of structural features required for the substrate affinity of GNA1. Structured summary: MINT:6700314: GNA1 (uniprotkb:Q96EK6) and GNA1 (uniprotkb:Q96EK6) bind (MI:0407) by X-ray crystallography (MI:0114) [Copyright &y& Elsevier]

The RlmL (YcbY) protein in Escherichia coli is an rRNA methyltransferase that is specific for m2G2445 modification of 23S RNA. The rlmL gene was cloned into the expression vector pET28a and expressed in the host E. coli strain BL21 (DE3). Recombinant protein with a six-histidine tag was purified by Ni2+-affinity chromatography followed by gel filtration. Crystals were grown using the hanging-drop vapour-diffusion method and a detergent was used as an additive to improve diffraction quality. The final crystals diffracted to 2.2 Å resolution. The crystals belonged to space group P21, with unit-cell parameters a = 73.6, b = 140.8, c = 102.9 Å, β = 102.3°. The crystal has a most probable solvent content of 62.8% with two molecules in the asymmetric unit. [ABSTRACT FROM AUTHOR]

Recent studies have indicated that Staphylococcus aureus can survive the nitrosative stress (caused by the radical nitric oxide; NO·) mounted by the immune system of the infected host. It does this by expressing a nitric oxide-inducible l-lactate dehydrogenase (Sa-LDH-1). Therefore, if efficient inhibitors of Sa-LDH-1 can be designed then Sa-LDH-1 could be a potential drug target against the pathogen S. aureus. For this purpose, the nitric acid-inducible LDH-1 from S. aureus COL strain has been cloned into the expression vector pET-28a(+) and the protein has been expressed, purified and crystallized. The Sa-LDH-1 crystal diffracted to 2.4 Å resolution at a home X-ray source and belonged to space group C2, with unit-cell parameters a = 131.4, b = 74.4, c = 103.2 Å, β = 133.4°. [ABSTRACT FROM AUTHOR]

The smu.412c gene encodes a putative histidine triad-like protein (SMU.412c) with 139 residues that is involved in cell-cycle regulation in Streptococcus mutans. The gene was cloned into the expression vector pET28a and subsequently expressed in Escherichia coli strain BL21 (DE3) to give a substantially soluble form of SMU.412c with a His6 tag at its N-terminus. The recombinant protein was purified to homogeneity in a two-step procedure involving Ni2+-chelating and size-exclusion chromatography. Crystals suitable for X-ray diffraction were obtained using the sitting-drop vapour-diffusion method and diffracted to 1.8 Å resolution on beamline BL6A at Photon Factory, Tsukuba, Japan. The crystal belonged to space group P41212, with unit-cell parameters a = b = 53.5, c = 141.1 Å. [ABSTRACT FROM AUTHOR]

The speA gene in Bacillus subtilis encodes arginine decarboxylase, which catalyzes the conversion of arginine to agmatine. Arginine decarboxylase is an important enzyme in polyamine metabolism in B. subtilis. In order to further illustrate the catalytic mechanism of arginine decarboxylase by determining the three-dimensional structure of the enzyme, the speA gene was amplified from B. subtilis genomic DNA and cloned into the expression vector pET-28a(+). SpeA was expressed in Escherichia coli and purified to homogeneity by nickel-chelation chromatography followed by size-exclusion chromatography. High-quality crystals were obtained using the hanging-drop vapour-diffusion method at 289 K. The best crystal diffracted to 2.0 Å resolution and belonged to space group P21, with unit-cell parameters a = 86.4, b = 63.3 c = 103.3 Å, β = 113.9°. [ABSTRACT FROM AUTHOR]

Summary: Streptococcus mutans is the primary etiological agent of dental caries in man and other mammalian organisms. This bacterium metabolizes carbohydrates actively and thrives under anaerobic conditions by fermenting l-ascorbate (Asc) via the sga operon, which includes SgaT, PtxB, and PtxA. These three proteins are members of the Asc family of enzyme II (EII) complexes of the bacterial phosphotransferase system. Here, we report the crystal structure of PtxB, solved by single-wavelength anomalous dispersion phasing, and that of PtxA, solved by molecular replacement, from S. mutans. PtxB provides the first crystal structure of an EIIB from the Asc family, composed of a central β sheet of parallel strands flanked by α helices on both sides. The structure of PtxB is similar to the structures of IIBMtl (IIB subunit of mannitol PTS) and IIBCel (IIB subunit of cellobiose) in Escherichia coli despite the low sequence identity. PtxA adopts a globular α/β sandwich structure. The phosphorylation-site His68 is situated between β2 and β3, within a hydrophobic pocket. We found that the hydrogen bond on Nδ1 of the active-site histidine is a common means of ensuring that phosphate is on the correct Nɛ2 site in many EIIA families. Finally, a model of the PtxB–PtxA complex was constructed, and a PtxA-phospho-PtxB state is proposed. Analyses of the two structures shed light on the catalytic mechanism of the phosphotransferase system. [Copyright &y& Elsevier]