| Fatty acid biosynthase (FAS) pathways divide into two distinct types, FAS I which is multidomain enzyme complex and found in mammals, and FAS II which consists of individual enzymes catalyzed sole reduction widely distribute in plants, bacteria and other microorganisms. The majority of the FAS II enzymes are essential for bacterial survival, and enoyl-ACP reductase (ENR) catalyzes the final and rate-limiting reaction in each round of the elongation cycle. If the activity of ENRs is inhibited, the growth of bacteria will be blocked. So, ENRs are usefull targets to develop new selectable antimicrobial agents. ENRs have four isozymes, named FabI, FabK, FabL and FabV. FabV is firstly discovered in Vibrio cholera recently. It is 60% larger than the typical SDR family members (which are generally about 250 amino acid residues in length), and the identity between FabV and other ENRs are very low. The spaces in the conservative catalytical motif of FabV between tyrosine and lysine residues are eight residues (Tyr-X8-Lys) which may be responsible for the resistance to triclosan. Therefrom, the fundamental differences of FabV and other ENRs support the proposal that FabV will becom a new target. As is known to all, bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most serious bacterial disease of rice in world. The results of sequences alignment indicate that FabV is the only ENR in Xoo, named XoFabV. Thus, XoFabV will be the sutible target to combat bacterial blight. However, any structure of FabV has not been resolved up to date. So, it is significative to figure out the structure and catalytic mechanism of FabV by developing related crystallographic studies.In this study, the Xoo fabV gene was cloned and expressed. The two-step purification by Ni2+-NTA affinity chromatography and size exclusion chromatography were developed to yield XoFabV proteins. Size exclusion chromatography and analytical ultracentrifugation proclaim that XoFabV has two forms, monomer and dimer, in solution, and the molecular weight of monomer is 51 kDa. The analysis of circular dichroism (CD) presents that the contents of a-helix andβ-strand have no difference between the monomer and dimer. Initial screening was performed based on the commercially available crystal screening kit from Hampton Research by the hanging drop vapor diffusion method. Optimizing the condition of crystal growth, we obtained some high quality crystals with reservoir solution containing 15% EPG3350,0.1 M Bis-Tris,125 mM NaCl (pH 5.5) at 16℃under the protein concentration of 10 mg/mL. We obtained the crystals of L-Se-Met substituted XoFabV from the same growth condition of native FabV as well. Crystals of L-SeMet substituted XoFabV were transferred to a reservoir solution with 15% (w/v) EPG3350 as a cryoprotectant for several seconds and flash-cooled in a cryostream of N2 gas at-73℃. A multiple-wavelength anomalous dispersion (MAD) data set was collected on the pharmaceutical industry beamline (BX41XU) at the Spring-8 synchrotron facility (Hyogo, Japan) using a Jupiter 210 CCD detector. All X-ray diffraction data diffracted to 1.67 A were integrated and scaled using the Crystalclear package (Rigaku). Diffraction data statistics are processed by PHENIX software package. The crystal of L-SeMet substituted XoFabV pertains to space group P212121, with a solvent content of 43.23% and unit-cell parameters a=49.250, b=73.350, c=106.550,α=β=γ=90.The BLASTP search results indicate that XoFabV belongs to the NAD (P) H-Rossmann superfamily. At the C-terminus, the conserved region is the motif FGFGRIDVDY (residues 377-386) binding FAD. A potential NADH-binding site GASSGYG is identified in residues 48-54 near the N-terminus of the sequence. A conservative catalytic motif YWHGAIGKAK is situated in residues 236-245. Superposition of the crystal structures of E. coli FabI monomer with NAD+and the XoFabV monomer demonstrates that there are significant differences between these enzymes, but each monomer has a classical Rossman fold. The Rossman fold of XoFabV contained sevenβ-strands (β1-β4,β9-β11) and seven a-helices (α2-α8). The parallelβ-sheet is flanked by helicesα2,α3 andα8, while on the other side flanked by helicesα4,α5, andα6. The long helix a5 contains a "kink" at residues G198 and G199. Via the analysis of XoFabV monomer, we infer that the NADH is bound to the enzyme on the shelf ofβ-sheet at the C-terminal end. Additionally, FAD binding site is located on the shelf formed byα1 andα10 out of the pocket. FAD might therefore stabilize the enzyme structure, but be not essential for catalysis. With the results, we can obtain the firsthand 3D-structure data of FabV which will become the theory foundation for the study of catalytic machanisms and screening of novel and effective compounds.
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