Crystal Structure And Inhibition Of OfChi-h,a Chitinase From The Insect Ostrinia Furnacalis

Pesticide innovation is crucial for pest control.The existing chemical pesticides can no longer meet the needs of pest control because of several defects,such as high toxicity and serious insecticide resistance.The basic reason for these problems is that the target site for pesticides is scarce.So,it is necessary for pesticide target innovation.Periodic chitin remodeling during insect growth and development requires three glycosyl hydrolase family 18(GH18)chitinases including group ? chitinase(Cht?),group ? chitinase(Cht?),and chitinase h(Chi-h).ChtI and Cht? shows highly conservative within species while Chi-h is exclusively distributed in lepidopteran insects,one of the most destructive crop pests.RNA interference of Chi-h resulted in molting deficiency and death indicating that Chi-h is indispensable for development and molting of insects.Thus,Chi-h can be a potential target for developing novel and eco-friendly agrochemicals.Until now,there is limited information about the structure-function relationship of Chi-h.Thus,the development of inhibitors for Chi-h is largely restricted.In this dissertation,we applied OfChi-h,a chitinase h derived from the agricultural pest Ostrinia furnacalis as representative of insect GH18 chitinase.By combining structural biology with biochemical characterization,the structure-function relationship of OfChi-h was revealed.A series of small molecule inhibitors targeting O/Chi-h were also developed.The main results in this dissertation are as follows:1.The structure-function relationship of OfChi-hHydrolytic activity assays demonstrated that OfChi-h showed higher activity toward the insoluble polymeric substrate than OfChtI.Hydrolytic pattern assays revealed that OfChi-h was an exo-chitinase that attack chitin from the reducing end.O/Chi-h and OfChtI work synergistically in chitin degradation when using insect cuticle chitin as substrate.Accord to the crystal structure of OfChi-h,OfChi-h was demonstrated to adopt a compact and elongated structure with two domains:a catalytic domain and a chitin binding domain.The catalytic domain consists of a(?/?)8-barrel and a chitinase insertion domain(CID).The CID deepens the substrate-binding cleft,which may enhance the binding affinity of OfChi-h to substrate.The chitin binding domain is located on the N-terminal of OfChi-h with aromatic residues on the surface.These residues may be involved in substrate binding.According to the OfChi-h-(GlcN)7 complex structure,OfChi-h was revealed to possesse a long and narrow substrate binding cleft with both two sides open.This cleft is composed of 9 lined-up aromatic residues whose distribution is highly asymmetric with regard to the enzymatic cleavage site.These aromatic residues play an important role in substrate binding via stacking interactions with sugar rings.Two extra ?-helices are found in the wall of the substrate-binding cleft.This unique structural element narrows the substrate-binding cleft,which may increase the binding affinity of OfChi-h for chitin chains and,thus,favor OfChi-h in hydrolyzing the crystalline substrate.2.The small molecular inhibitors of OfChi-h(1)Berberine and its analogsB erberine was reported to a broad-spectrum inhibitor of various GH18 chitinases including OfChi-h with only moderate Ki values.It inhibited GH18 chitinases mainly via ?-? stacking interactions between the tetracycle plane and the aromatic residues in the binding pocket.With the conjugate plane of berberine retained,a modification of 9-O-position resulted in significant improvement of inhibitory activity and selectivity of berberine by enhancing its binding affinities toward OfChi-h.The analog 4c with 6-fluoronicotinoyl substituent group showed optimal inhibitory activity toward OfChi-h with a Ki value of 80 nM.(2)Substrate analoguesThe substrate analogues TMG-(GlcNAc)4 showed selective inhibitory activities against OiChi-h.Injection of TMG-(GlcNAc)4 into O.furnacalis larvae led to severe defects in pupation.Hydrolytic products assay revealed that TMG-(GlcNAc)4 can be further degraded into TMG-(GlcNAc)2 and TMG-GlcNAc by OfChi-h.Since TMG-(GlcNAc)2 showed high inhibitory activity toward O/Chi-h as well,we deduce that TMG-(GlcNAc)2 is the final stable inhibitor of OfChi-h.(3)Microbial secondary metabolite phlegmacin B1By screening a library of microbial secondary metabolites,phlegmacin B1 was found to be the competitive inhibitor of both GH18 chitinase OfChi-h and GH2O?-N-acetylhexosaminidase OfHexl with Ki values of 5.5 ?M and 26 ?M,respectively.It was predicted to bind to the active pockets of both OfChi-h and OfHexl via hydrophobic and hydrogen-bond interactions by using molecular docking and molecular dynamics simulations.Injection and feeding experiments demonstrated that phlegmacin Bi has insecticidal effect on O.furnacalis larvae.In summary,the structure-function relationship of OfChi-h was revealed in this dissertation,which is in favor of understanding the insect chitin degradation system.What's more,a series of small molecule inhibitors targeting OfChi-h were obtained,which provides new scaffold for the development of green pesticides.This work is of great significance for insect-mediated disease therapy and agricultural pest control.