My laboratory has been using the macromolecular crystallography beamlines BL-6A, BL-6B, and BL-18B at Photon Factory since early 1990s to collect high-resolution X-ray diffraction data. Photon Factory data were used to solve a number of protein structures, including Bacillus licheniformis a-amylase, sweet potato P-amylase, Pseudomonas cepacia lipase, Pseudomonas fluorescens carboxylesterase, xylose isomerases from Thermus caldophilus and Thermus thermophilus, Saccharomyces cerevisiae Ypd1p, Methanococcus jannaschii malatenactate dehydrogenase, deoxycytidylate hydroxymethylase from bacteriophage T4, rice non-specific lipid transfer protein, ecotin from Escherichia coli, barley chitinase, DNA polymerase I from Thermus aquaticus, and DNA ligase from Thermus filiformis. Two of these structures will be described briefly in this article.
In this paper, a scan driver with extra clock signal using amorphous indium‐gallium‐zinc‐oxide (a‐InGaZnO) thin‐film transistors (TFTs) is proposed. Extra clock signal with variable frequency modulates the pull‐down unit of scan driver in order to reduce the power consumption. Proposed scan driver is suitable for operating extreme low‐power displays such as always on display.
Enoyl-ACP reductase (ENR; EC 1.3.1.9), encoded by the fabI gene, is a key enzyme of the Type II fatty-acid biosynthetic system in prokaryotes and plants. It uses NADH or NADPH as the cofactor to reduce the double bond between C2 and C3 positions of a fatty acyl chain bound to the acyl carrier protein in the terminal rate-limiting step of the fatty acid chain elongation cycle.1 Because it shows low overall sequence homology with mammalian enzymes, it is a potential target for antibacterial discovery. Two classes of ENR inhibitors are already used in the clinic (isoniazid) and in the consumer products (triclosan), and other classes of novel ENR inhibitors are being developed. Several classes of ENR inhibitors have been structurally characterized as complexes with their target enzymes. Crystal structures of Escherichia coli ENR (also called EnvM) complexed with diazaborines2 and triclosan,3-5 Mycobacterium tuberculosis ENR (also called InhA) complexed with isoniazid,6, 7 Brassica napus ENR complexed with triclosan,8 and Plasmodium falciparum ENR complexed with triclosan and its analogs9-11 have been reported. Diazaborines and the activated form of isoniazid are attached covalently to either the nicotinamide ring or the 2′-hydroxyl of the nicotinamide ribose of NADH.2, 6 In comparison, triclosan binds noncovalently adjacent to the bound cofactor.11 Triclosan (2,4,4′-trichlolo-2′-hydroxydiphenyl ether) is a bactericidal agent and is effective against a variety of microorganisms, including Gram-positive and Gram-negative pathogenic bacteria as well as fungi and yeasts.4 It is used in the manufacture of commercial products, including clothing, materials for food processing, personal care products (e.g., soaps and toothpaste), and surgical items (e.g., sutures).12 The release of triclosan into the environment is of particular concern as it is structurally similar to thyroid hormone and may, therefore, represent a potential disruptor of normal growth and development in wildlife and humans that involve thyroid hormone action.13 Therefore, it is desirable to develop ENR inhibitors that are safer than triclosan. Helicobacter pylori is a Gram-negative bacterium responsible for gastritis, peptic ulcer, and gastric cancer.14 Because of its importance as one of the major human pathogens, structural information on H. pylori ENR is important for structure-based design of triclosan analogs against H. pylori. Here we present the crystal structures of H. pylori ENR as ternary complexes with NAD+ and triclosan (or its analog diclosan (4,4′-dichlolo-2-hydroxydiphenyl ether)). As structural data on the diclosan complex has not been reported previously, this study complements the existing structural data on ENRs in designing triclosan derivatives as ENR inhibitors. Overexpression, crystallization, and X-ray data collection of H. pylori ENR were reported previously.15 We solved the structure of H. pylori ENR by the molecular replacement method using the model of E. coli ENR2 (Protein Data Bank ID code 1DFI) as a search model. Cross-rotation search followed by translation search was performed using the program CNS.16 Subsequent manual model building was done using the program O.17 The model was refined with the program CNS and several rounds of model building, simulated annealing, positional refinement, and individual B-factor refinement were performed. We refined two structures: (i) the ternary complex with triclosan and NAD+, and (ii) the ternary complex with diclosan and NAD+. The former model has been refined to crystallographic Rwork and Rfree values of 23.3% and 25.9% for the resolution range of 20 Å–2.5 Å, while the latter model has been refined to 22.4% and 24.9% for the resolution range of 29 Å–2.3 Å, respectively. Each model accounts for 1096 amino acid residues of the tetrameric ENR (residues 2–275), four NAD+ cofactors, four triclosan (or diclosan) molecules, and 214 (or 218) water molecules in each asymmetric unit. Only the amino terminal methionine is missing from the model. Refinement statistics are shown in Table I. Atomic coordinates of the ternary complex of H. pylori ENR with NAD+ and triclosan have been deposited into the Protein Data Bank as 2PD3, and the ternary complex of ENR with NAD+ and diclosan as 2PD4. We have determined the crystal structure of H. pylori ENR complexed either with triclosan or with diclosan [Fig. 1(A)]. H. pylori ENR is a homotetramer, like other ENRs. Four independent subunits of a H. pylori ENR homotetramer in the asymmetric unit adopt similar conformations [Fig. 1(B)]. When we compare subunit A against other subunits B–D for both models, the root mean square (r.m.s.) deviations range between 0.1 Å and 0.2 Å for 274 Cα atom pairs. The monomer of H. pylori ENR has a Rossman fold, similar to other ENR structures. When we structurally compare H. pylori ENR (subunit A of the triclosan complex) with ENRs from E. coli (PDB ID code 1C14),5 M. tuberculosis (PDB ID code 1P45),7 B. napus (PDB ID code 1D7O),8 and P. falciparum (PDB ID code 1UH5),11 the r.m.s. difference is 0.8 Å (for 255 Cα atom pairs of structurally aligned residues), 1.7 Å (for 250 Cα atom pairs), 1.6 Å (for 247 Cα atom pairs), and 1.5 Å (for 247 Cα atom pairs), respectively. No significant structural differences are observed between triclosan and diclosan complexes of H. pylori ENR, with the r.m.s. difference being 0.25 Å for 274 Cα atom pairs (for subunit A). Monomer and tetramer structures of H. pylori ENR and electron density maps of the bound ligands. (A) Monomer structure. Secondary structure elements were defined by PyMOL (DeLano, 2002, The PyMOL Molecular Graphics System, http://pymol.sourceforge.net/). NAD+ and triclosan bound to the active site of subunit A are shown as ball-and-stick models. (B) Tetrameric architecture. NAD+ and triclosan are shown. (C) 2Fo−Fc electron density maps of the bound ligands, superimposed on triclosan (subunit A), diclosan (subunit A), and NAD+ (subunit A of the triclosan complex). (D) Superposition of triclosan (cyan) and diclosan (purple). Phenolic rings (ring A) of triclosan and diclosan are superimposed. In the H. pylori ENR-NAD+-triclosan ternary complex, triclosan is bound in a similar manner to other ENRs [Fig. 2(A)]. We have also determined the structure of the ENR-NAD+-diclosan ternary complex. The binding mode of diclosan is highly similar to that of triclosan. The absence of a chlorine atom at the atom position 2′ of ring B in diclosan has virtually no effect on the ligand binding (Fig. 2). Triclosan (or diclosan) is positioned adjacent to NAD+ in a hydrophobic pocket formed by hydrophobic residues (Phe94, Leu100, Tyr155, Ala195, Ile199, and Phe202) (Fig. 2). Ala195, Ile199, and Phe202 are part of the so-called substrate-binding loop.11 The phenolic ring (ring A) of triclosan (or diclosan) makes π–π stacking interactions with the nicotinamide ring of the cofactor [Fig. 2(B,C)]. A conserved hydrogen-bonding network is formed between the 2′-hydroxyl group of triclosan (or diclosan), the side chain of Tyr155, and the nicotinamide ribose of NAD+ [Fig. 2(B,C)]. When we superimpose the two phenolic rings of triclosan and diclosan, the 4′-chlorophenoxy ring of diclosan makes an angle of ∼15° to the 2,4-dichlorophenoxy ring of triclosan for all four subunits [ring B in Fig. 1(D)]. Binding mode of triclosan, diclosan, and NAD+. (A) Superposition of H. pylori ENR (subunit A of the triclosan complex, colored in red) with ENRs from E. coli (PDB ID code 1C14, colored in yellow), M. tuberculosis (PDB ID code 1P45, colored in pink), B. napus (PDB ID code 1D7O, colored in cyan), and P. falciparum (PDB ID code 1UH5, colored in green) ENRs (monomer A). (B) Binding site of triclosan and NAD+ in subunit A. (C) Binding site of diclosan and NAD+ in subunit A. The conformation of a flexible substrate-binding loop was suggested to be the major determinant for different triclosan-binding affinities by different ENRs.11 The substrate-binding loop of H. pylori ENR containing Ala195, Ile199, and Phe202 has a "closed" conformation [Fig. 2(A)], which is similar to that of E. coli and P. falciparum ENRs. The corresponding loop in M. tuberculosis ENR has an intermediate conformation between the "closed" conformation and the "open" conformation of B. napus ENR.11 The side chains of Ile199 and Phe202 of H. pylori ENR are positioned slightly closer to triclosan than the corresponding residues of E. coli and P. falciparum ENRs [Fig. 2(A)]. Recently, a series of triclosan derivatives were synthesized and their inhibitory activities were assessed.18 The present structures of H. pylori ENR in complex with either triclosan or diclosan would be useful in designing ENR inhibitors. We thank the beamline staffs for assistance during synchrotron data collection (BL-6B of Pohang Light Source, Korea and BL-18B of Photon Factory, Japan).
Lipid A, a constituent of lipopolysaccharides, is essential for the growth and virulence of most Gram-negative bacteria. This makes its biosynthetic enzymes potential targets for development of new antibacterial agents. The first step of lipid A biosynthesis is catalyzed by the enzyme UDP-N-acetylglucosamine acyltransferase (LpxA). LpxA from the pathogenic bacterium Helicobacter pylori has been overexpressed in Escherichia coli and crystallized at 297 K using ammonium sulfate and sodium/potassium tartrate as precipitants in the presence of a detergent. Diffraction data to 2.1 Å resolution have been collected from a native crystal. The crystal belongs to space group P6322, with unit-cell parameters a = b = 90.69, c = 148.20 Å. The asymmetric unit contains one subunit of LpxA, with a crystal volume per protein mass (VM) of 2.87 Å3 Da−1 and a solvent content of 57.1%.
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