| Babesiosis，which transmitted by ticks and parasitizes in the erythrocytes of mammals, also calledtick fever or piroplasmosis, are the second most common blood-borne parasites of mammals after thetrypanosomes.and impacted strongly on the livestock industry and associated massive economic costs intropical and subtropical areas. Currently, there is no effective recombinant vaccine againstapicomplexan pathogens including Babesia. Although some compounds are available to treat babesiosis,the development of drug resistance, reinfection, side effects, and residue problems are likely events.Therefore, there is a need to develop novel chemotherapeutic agents against these pathogens for theveterinary market. The MEP pathway in apicoplast of the Babeisa bovis is vital for the parasites. Theisoprenoid mevalonate independent biosynthesis of parasitic protozoa in the apicoplast is a promisingchemotherapeutic target, because this pathway is different from the mevalonate pathway in mammalsand is essential to such parasites. Enzyme genes associated with MEP pathway in B.bovis Shannxianwere cloned and studied especially the DXS.1. Seven genes associated with MEP pathway were cloned using the cDNA of B.bovis Shannxianisolated as template, and analyzed the sequences by BLAST. Results shows that full length of DXSgene was2061bp, encoding686amino acid, the similarity with B.bovis T2Bo in GenBank was98.0%.The other six genes were patial sequences, including dxr（GenBank ID：）；IspC（GenBank ID：）；IspD（GenBank ID：KF939613）；IspE（GenBank ID：）；IspF（GenBank ID：）；IspG（GenBank ID：），the similarity between deduced amino acid sequences and the B.bovis T2Bo in GenBank were greaterthan90%. These datas might be of great help in gaining a better understanding of those genes.2. For targeted disruption of a B.bovis gene, we attempted to construct the plasmid of knockoutthe dxs gene by homologous recombination. To generate the targeting construct (DHFR-gfp-DXS KO),5’ and3’ portions of dxs were cloned into the DHFR-gfp plasmid. The eukaryotic expression dxs genewith EGFP fluorescent reporter gene and the prokaryotic expression dxs gene with6*His tag wereconstructed. Those constucted were provided the materials for studing the functional and characterize ofDXS further.3. The BbDXS expression condition were optimized. The rBbDXS were expression with finalconcentration0.6mM IPTG at18℃and purified by affinity chromatography. Steady-state kineticconstants for rBbDXS calculated by HPLC-MS/MS from the initial velocities measured at varyingconcentrations of substrates were as follows: Kpyrm=790±52μM，Kcat=7.4S-1；KGAPm=380±46μM，Kcat=10.2S-1。4. The six site mutant BbDXS plasmid were constructed based the pET-DXS, and site mutantBbDXS protein were expressed and purified using the same condition. Product of the catalytic reactionof site mutant BbDXS were measured using the same methods. The results showed that Asp209，Ile415，Asp474were crucial for catalysis. The Asp209, Ile415, Asp474may be important for the activation ofthe coenzyme or substrate because their side chains interact with each other and with TPP or substrate.In comparison, mutations of Ser178, Gly210, Phe442had minimal impact on the catalytic activity. 5. EcDXS and the pyruvate and inhibitor ketoclomazone were docked using the molecular dockingsoftware. The docking results showed that pyruvate could have interactions with the side chains ofGlu348，Gly349. There may be two situation using ketoclomazone docking with the EcDXS. One is theketocolmazone may loated the same pocket with the GAP and interaction with the side chains of Arg99,Arg478. The other is the ketocolmazone may loated the same pocket with the pyruvate and interactionwith the side chains of Arg347, Glu348, Gly349. Therefore, the ketoclomazone may inhibit thesubstrates combine with enzyme. The coenzyme TPP and substrate pyruvate were docking with thehomology modeling BbDXS using the molecular docking software. The results showed that Phe442,Tyr439, Gly176, Ser178, Gly210and Ser211are crucial for coenzyme TPP combination. The dockingresults showed that pyruvate could have interctions with the side chains of Asn236, Gly208, Gln136inBbDXS.6. The pharmacophore model were derived based on the DXS inhibitors using the Discovery studio3.5. We carried out drug screening in the DrugDivrse database using the optimal pharmacophore model5.2518moleculars were obtained and160of them the FitValue were greater than3. The160molecularswere docked with the EcDXS, and46moleculars which combined better with EcDXS were obtained.These datas might be of great help in studying the inhibitor of DXS further. |
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