Crystal Structures Analysis And Catalytic Mechanism Of Babesia Lactate Dehudrogenase

Human babesiosis is caused by apicomplexan Babesia parasites,including B.microti,B.duncani,B.crassa,B.sp.MOI,B.divergens and B.venatorum.Among them,B.microti is the most common-cause of human and rodent babesiosis,and is frequently accompanied by high parasitemia.No available vaccine is recommended and drugs for treatment of the disease are associated with high failure rate and side effect.As B.microti lacks a traditional TCA cycle and dominatingly relies on anaerobic metabolism to produce ATP,we suspect that B.microti lactate dehydrogenase（Bm LDH）,a key glycolytic enzyme,plays a critical role in B.microti ATP supply,and may be a drug target for aborting the energy supply of B.microti to inhibit the proliferation of the parasite in RBCs.（1）Biological function identification.Both gossypol and oxamate exhibited significant effects on B.microti in vitro in the range of micromol,and the gossypol also inhibited the growth of B.microti in vivo.The c DNA of B.microti and B.orientalis were used as templates to clone the full-length ORFs of Bm LDH and Bo LDH,which was inserted into p ET-28a vector,and the sequencing plasmids were transformed into E.coli BL21.The r Bm LDH and r Bo LDH had a good immunogenicity and can distinguish Babesia-infected positive serum and uninfected negative serum.The natvie Bm LDH and Bo LDH were about 37 k Da size,and mainly located in the cytoplasm.Finally,the enzyme kinetics parameters of Bm LDH,such as K_M,K_cat,and V_max,were extracted by varying substrate or cofactor concentrations,and the inhibition constant K_iof gossypol and oxamate on Bm LDH were determined as 0.67 and 65.92μM,respectively.（2）Crystal structure analysis.The purified r Bm LDH and r Bo LDH separately were filtered by molecular sieve chromatography（peak time of 60 min）.The r Bm LDH and r Bo LDH was concentrated,and further used for the initial screening of crystallization conditions.By optimizing crystallization conditions,Bm LDH apo form,Bo LDH apo form and Bm LDH complex with NADH and oxamate were successfully obtained with high qualities.The structure data of Bm LDH apo and co-crystal were collected by X-ray diffraction,and the structures of apo form and co-crystal form were determined at 2.79?and 1.89?resolution.Structural comparison indicated that the overall structure of Bm LDH shares a conserved feature with that of human and plasmodium LDHs.In addition,these structures revealed that the active pocket of Bm LDH undergoes a major conformational change from an opened and disordered to a closed and stabilized state,when the co-factor and substrate binds into.（3）Identification and verification of catalytic activity switches.Based on the Bm LDH complex structure,ten residues that interact with NADH in the catalytic center were found,and seven of them were mutated.The result of enzyme activity experiment showed that G97A,R99A and N138A significantly reduced the catalytic activity of Bm LDH by more than 86%.Further,we explored the difference between Bm LDH and human LDH-A catalytic key sites,the three amino acid residues in human LDH-A were mutated,and the result displyed that the catalytic activities of human LDH-A were significantly reduced by Gly97A and N138A,while the catalytic activitie were not affected by R99A.By co-crystallization of Bm LDH^R99A,SPR and ITC experiments,we elucidated the molecular mechanism of the inactivation of Bm LDH^R99A mutant.Together these results indicated that the R99A mutation not affects both the overall structure and the affinity to NADH of Bm LDH,but the closure of the Bm LDH catalytic center.（4）Molecular docking and virtual screening.Base on the Bm LDH crystal structure,13kinds of high score compounds were acquired from a 150,000 drug library,and the three derivatives of gossypol（NDCA,DBHCA and DHNA）were selected fot activity tests,the results show that the compound DBHCA and DHNA at micromole level inhibited both the Bm LDH catalytic activity and B.microti in vitro.The MTT experimentes further demonstrated that the compound DHNA had low poisonous to the Vero cells（SI>25）.Furthermore,the affinity（K_D value）and pharmacokine binding and dissociation constants(K_onand K_off)of the two compounds to Bm LDH were determined by SPR experiments.In conlusion,we cloned and expressed the Bm LDH gene,characterized the immunogenicity and localization of Bm LDH,and determined the enzyme kinetics curves of Bm LDH.Two LDH inhibitors gossypol and oxamate inhibited both the catalytic activity of Bm LDH and the growth of B.microti in vitro.Further,the crystals of the apo Bm LDH and the complex state with NADH and oxamate revealed the Bm LDH catalytic mechanism and the key catalytic residues.By virtual screening and molecular docking,a series of potential Bm LDH inhibitors were acquired.Subsequently,we evaluated two kinds of new compounds against the Bm LDH catalytic activity and in vitro the growth of B.microti.Together these structural and biochemical data highlight significant differences between B.microti and human LDH enzymes and suggest that Bm LDH could be a suitable target for the development of selective anti-babesial inhibitors.