Protein Expression, Purification And Crystallographic Analysis Of β-hydroxyacyl-ACP Dehydratase(FabZ) Fron Candidatus Liberobacter Asiaticum

Citrus Huanglongbing (HLB) is caused by gram-negative pathogenic bacterium and spread by Citrus psyllid, that causes citrus to change to yellow and oddly shaped, thus affecting citrus production. There are no effective prevention and control measures for citrus Huanglongbing. Nowadays, the outbreaks of infection diseases caused by pathogenic microorganisms, so novel antibacterial agents acting on new targets are needed urgently. Fatty acid biosynthesis is very important for all living cell. In general, Fatty acid biosynthesis is classified into two different pathways (FAS I and FAS II) because of the architecture of the enzyme involved. In FAS I pathway, the involved synthases found in mammals and fungi are large multifunctional enzymes with multiple domains that catalyze various reactions of FAS. In contrast, the FAS for plant chloroplasts and bacteria belongs to the FAS II pathway, an individual enzyme catalyzes every step of biosynthesis, and they are always highly specificity in different bacteria. The (3-hydroxyacyl-ACP dehydratase (FabZ) catalyzes an essential step in the FAS II pathway. It catalyzes the dehydration of β-hydrozyacyl-ACP to trans-2-acyl-ACP. If β-hydroxyacyl-ACP dehydratase is inhibited, the growth and reproduction will be all blocking. So β-hydroxyacyl-ACP dehydratase is new important targets for novel antibacterial.In our article, according to clone, expression and purification, we get the ClFabZ that molecular weight of monomer is18kDa. The size exclusion chromatography proved that the ClFabZ protein is being hexamer in solution. Using sitting drop vapor-liquid diffusion method and screening through the use of the United States Hampton Research crystal screening kit, we initially obtain the crystsllization conditions of ClFabZ. Then we optimize this condition and get the optimum crystallization conditions of2%v/v Tacsimate pH5.0,0.1M Sodium citrate tribasic dihydrate pH5.6,16%w/v Polyethylene glycol3350at20℃with the protein concentration of7mg/mL. The crystal data is obtained by culture under the same conditions as above L-Se-Met substituted ClFabZ. The X-ray diffraction data was collected on shanghai Synchrotron Radiation, it has a2.9A resolution. The crystal data are processed by PHENIX software. The crystal of L-Se-Met substituted ClFabZ belongs to space group P6122, unit-cell parameters was a=b=75.346, c=353.236, α=β=90,γ=120.The BLASTP search results indicate that the structure of the ClFabZ dimer is similar to other known FabZ structures. The monomer structure of ClFabZ adopts a typical mixed β+α "hot dog" fold. Each subunit consists of eight-stranded highly curved anti-parallel (3-sheet that wraps around a central a-helix a3. Two short a-helix are formed at the N terminus, one is at the beginning of the N terminus and the other is between β2and α3. The active sites of ClFabZ were defined by analysis of other known FabZ structures. It may reside on the subunit-subunit interface, and thus each dimer harbors two active sites. The catalyze active site may be conserved His57and Glu71’(indicates residue from the other subunit of the dimer) by analysis of other known FabZ structures, resides in the middle of the tunnel.With the results of the crystal structure, we can not only obtain the spatial structure data of FabZ, but also provide a basis for designing type-selective drugs by targeting FabZ. We also can provide the important theoretical basis for the catalytic mechanism of FabZ.