Molecular And Biochemical Characterization β-Hydroxyacyl-Acyl Carrier Protein Dehydratase(Had)of Eimeria Tenella

Avian coccidiosis is an intestinal parasitic disease which is caused by the apicomplexan parasites of Genus Eimeria hosted in the intestinal epithelial cells of chicken, and may cause enormous economic loss of poultry industry. The strategies for controlling this economically important disease rely heavily on chemoprophylaxis during the past decades. Due to the increasing emergency of drug-resistant strains, the man have to explore novel targets for new anticoccidial development. The type Ⅱ Fatty acid synthesis pathway (FAS Ⅱ) is a special plant-like metabolic pathway localized in the organelle apicoplast of Eimeria spp and other apicomplexans. In this pathway the initial substrate acetyl-CoA is converted into the end product palmitic acid under the catalysis of7enzymes.In this study, we focus on the molecular and biochemical characterization of the third enzyme in this novel biosynthetic pathway, β-hydroxyacyl-ACP dehydratase (HAD), which catalyzes the dehydration of β-hydroxyacyl-ACP into trans-2-enoyl-ACP. Firstly, the gene (ORF) encoding the HAD of Eimeria tenella (EtHAD) was assemblied and predicted by the data-mining and annotating from the Eimeria tenella genome database (EtGeneDB, http://www.sanger.ac.uk/Projects/E_tenella/) based on the Toxoplasm gondii(TgFabZ), Plasmodium falciparum(PfFabZ) and other bacterial HAD. According to the predicted gene sequence, the specific primers were designed. The partial sequence fragment of EtHAD gene coding the maturing enzyme protein was amplified by RT-PCR from E. tenella total RNA template.This novel sequence encoded a protein composed with160amino acid. The multiple alignment results reveal that the amino acid sequence of EtHAD is shared identities of30-70%with those HADs of other aoicpmplexans and bacteria. In addition, five highly conserved motif ("PHRYPFLLVD"、"GHFP"、"PGVL"、"EALAQ"and"PGD") and two active sites were observed.Since there is no any effective orthology for predicting the apicoplast-target transit peptide, the potential transit peptide in EtHAD aa sequence was probed using the alignment by the ClustalW (v2.1.1). The ORF fragment coding the potential maturing EtHAD protein was cloned and inserted into pET-32a(+) vector. After transformation into expression host bacteria E. coli Rosseta(DE3). The bacteria were grown at37℃in Luria Broth until OD600of0.6, and add1mM isopropyl-β-D-thiogalactopyranoside to induce the recombinant expression. the results of electrophoresis revealed the recombinant EtHAD is rich in supernatant of bacteria culture.The recombinant enzyme was purified by, single step affinity chromatography using Ni-NTA column.To determine the subcellar localization of EtHAD in the parasites, the specificial polyclonal antibodies against EtHAD were produced using BALB/c mice. The imaging visualization by the multiple-fluorscent staining revealed that the EtHAD was located in the apicoplast of E. tenella sporozoites under the confocal microscope.The enzymatic activity of EtHAD was determined using the crotonoyl-CoA as substrate. The conversion of crotonoyl-CoA to P-hydoxybutyryl-CoA was monitored following the decrease of absorbance value at260nm.The results suggested that the purified TRX-EtHAD was active with a Km value of79.4±2.96(μM and Vmax value of0.06985nM/min/μg.(-)-catechin gallates and Juglone was effective to inhibit EtHAD. Biochemical analysis of inhibitory kinetics revealed these two compounds can inhibit this novel Eimerian enzyme with IC50of10.5±0.6μM and35.4±1.OμM, respectively. Our primary results suggested that this enzyme may be explored as a potential and novel drug target.