Study On Kynurenine Formamidase And Inhibitor In Homo Sapiens

The kynurenine pathway(KP)is one of the main catabolic pathways of L-tryptophan.The metabolic intermediate of KP are associated with inflammation and excitatory neurotransmitters.The disorder of KP might lead to inflammatory diseases of the nervous system,such as Alzheimer's disease,Huntington's disease and amyotrophic lateral cord neurosis.KP is considered to play a crucial role in immunosuppression and tumor induction tolerance.Inhibiting the enzymes of KP could be a new method for the treatment of neurological disorders and tumors.Kynurenine formamidase(KFase;EC 3.5.19)is one of the rate-limiting enzymes in KP,KFase may be a new therapeutic target for neurological disorders and cancer.The catalytic triad of KFase is constructed by the residues of Ser,Asp and His.Virtual screening for KFase inhibitors might contribute to the candidate or precursors study for drugs which used for treatment of neurological disorders and tumors.In this study,a three-dimensional structure model of Homo sapiens KFase was constructed by the crystal structure of Drosophila melanogaster KFase(PDB ID:4E11),and the three-dimensional structure characteristics were analyzed.Molecular docking was performed to obtain the complex model of Homo sapiens KFase and the substrate N-formyl-L-kynurenine(NFK).The interaction between the enzyme and substrate was analyzed by molecular dynamics simulation.Virtual screening was used to screen inhibitors of KFase from the small molecule database ZINC and PubChem.The gene KFA of human KFase was cloned from HepG2 cells,and construct the pTYB12 recombinant plasmid containing the gene.Fusion protein expressed in E.coli BL21(DE3)was purified by chitin beads.The purified protein was used for the activity and kinetics analysis.The three-dimensional structure model of human KFase indicated that the catalytic triad of Ser164,Asp244 and His279 are located at the bottom of the active clave,and the space of active clave is big enough to accommodate NFK.The formamide group of NKF closed to the residue Ser164 of the active site,and the residue Tyr45 might participate in stabilizing the NFK at the active clave by the analysis of molecular docking.The results of molecular dynamics simulation shown that the residues Ser164,Asp244 and His279 were stable in the simulation process,and the Tyr45 has a larger range of movement,which is fit the structure requirement of the enzyme for catalyzed reaction.Virtual screening has obtained 9 small molecule compounds.KFase was successfully expressed in prokaryotic E.coli BL21(DE3).The computer simulation of the three-dimensional structure,expression and purification of human KFase might useful for studying the three-dimensional structure,physiological and biochemical characteristics.Virtual screening of small molecule inhibitors will provide candidate or precursor for the inhibitors of KFase,and provide a novel therapeutic methods for the treatment of variety diseases.In addition,we obtained KFase with catalytic activity,laying a foundation for the study of subsequent inhibitors.