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The fatty acid biosynthetic pathway is divided into two types, i.e. FAS I and FAS II. Wherein FAS I in a series of enzyme active site is located on a different domain of a large multifunctional composite body, such as a mammal; FAS II in each of the activity sites in the type of protein, each reaction It is widely present in plants, bacteria and other organisms by a separate enzyme catalysis. Enoyl-ACP reductase (enoyl-ACP reductase, ENR) is the catalytic bacterial FAS II key enzyme in the de novo synthesis pathway final step in the reduction reaction. If enoyl-ACP reductase is inhibited, the growth and reproduction of bacteria will be blocked. Therefore, enoyl-ACP reductase is a development of new antibacterial agents is a very useful target. Enoyl-ACP reductase the four isozyme, i.e. of FabI, FabK, FabL, FabV, distribution in the different species of bacteria. Where FabV the recently found from Vibrio cholera (Vibrio Cholera), from a structural point of view, than ordinary members of the sequence of the short-chain dehydrogenase / reductase superfamily (short-chain dehydrogenase / reductase, SDR) 60% longer, and the low sequence identity with other members of the family ENRs. Conserved catalytic motifs, tyrosine and lysine in the sequence at intervals of 8 amino acids (Tyr-X8-Lys), and 1-2 more than the other members of the the ENRs family family, which may be the FabV suppression against a broad spectrum agents - triclosan (Triclosan) is the main reason for resistance. The result, FabV expected to become a new target for screening potential selective inhibitor. Rice bacterial blight is the most serious bacterial diseases of rice, caused by Xanthomonas bacteria (Xanthomonas oryzae), the X. oryzae only the newly discovered FabV (XoFabV). Homology of the FabV not currently exist, which its targets inhibitor screening and catalytic mechanism research difficult. Crystallography for the first time XoFabV three-dimensional structure for reveal the FabV the structure and catalytic mechanism will be very meaningful. This issue through the cloning, expression, purification XoFabV protein, with a molecular exclusion chromatography analysis found its solution in presence of two trimer form, both determined according to the results of analytical ultracentrifugation trimer were in the form of monomers and dimer, the molecular weight of the monomer is approximately 51 kDa. The similarities and differences of the secondary structure of the monomeric and dimeric forms of circular dichroism studies found that both secondary structure are rich in alpha-helices and beta-fold, and the content of the two no difference. Using the hanging drop vapor-liquid diffusion method, and the United States Hampton Research crystal screening kit for screening to obtain the crystallization conditions XoFabV and optimized to obtain the optimum crystallization conditions for 15% PEG3350, 0.1 M Bis-Tris pH 5.5 , 125 mM NaCl, the protein concentration of 10mg/mL, optimum growth temperature of 16 ° C. Then culture to obtain high-quality of selenomethionine XoFabV crystal in the same conditions as above, by the Japan Spring-8 synchrotron radiation meter the BL41XU light source collection of multi-wavelength anomalous diffraction data with a resolution of up to 1.67A. Among them, the analytical parameters to -73 ° C, the wavelength of the light source for the Peak: 0.9798 A: EDGE: 0.9801 A: Remote: 0.9000 A. Then the resulting crystal data were analyzed using the PHENIX package display XoFabV belongs to space group P212121, with unit cell parameters a = 49.250, b = 73.350, c = 106.550, alpha = beta = gamma = 90 °, the solvent content of 43.23% . According to the amino acid sequence analysis revealed that ordinary SDR family conserved motifs YX3-7K than the results, the XoFabV and slender Euglena (Euglena gracilis) enoyl-CoA reductase (TER) of the amino acid sequence similarity. XoFabV conserved catalytic motifs YX8K located at 236-245, 48-54 in the N-terminus containing medium-chain dehydrogenation reductase superfamily (middle-chain dehydrogenase / reductase, MDR) superfamily NADH typical binding sites GX1-3GX1-3G, i.e. rich the Gly GASSGYG motifs. Containing FAD in the C-terminal 377-386 binding sites FGFXXXXXDY and i.e. FGFGRIDVDY. XoFabV structure in addition to having a similarity with the E. coli enoyl-ACP the reductase (EcFabI) ??Rossman folded redundant portion of its sequence is also formed a giant hat Structure (under the presence of catalytic sites) and FAD binding region (located on the outside of the pocket). Rossman fold in XoFabV structure consists of seven the flank alpha-helix (a2-α8) and 7 beta-folding (beta1-beta4, β9-β11) formed in the p-pleated sheet. , Alpha2, alpha3, α8 is located in the side of the layer of the beta-pleated sheet, α4, α5, α6 is located at the other side, the long spiral by alpha5 within G198, G199 at a \NADH binding sites may be located in the beta-sheet layer on the stent, and substrate binding sites should be located at the top of the binding sites of the NADH. At the same time, FAD binding sites are located outside of the pocket, α1, α10 bracket speculated that the primary role of the FAD binding is stable XoFabV the conformation. Get XoFabV crystal structure, first-hand provide FabV the spatial structure of data, but also provide a theoretical basis for the FabV targeting new efficient selective inhibitor design with screening and FabV of the catalytic mechanism of.
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