| Glycoside hydrolases(GHs),which are widely distributed in nature,are enzymes participate in the hydrolysis of glycosidic bonds in complex sugars.The GH18 chitinases and GH84 O-β-N-acetylglucosaminidases are two important types of glycoside hydrolases,they both adopt the substrate-assisted retaining mechanism.GH18 chitinases are extremely common enzymes in organisms including bacterium,fungi,nematodes,plants,arthropods and mammals.They are responsible for degrading chitin in the environment or inside the organism and play important roles in many physiological activities including cell nutrition,pathogen invasion,embryonic development.insect molting and immune defense.For insects.chitin is a major structural component of the exoskeleton,trachea and peritrophic membrane,so chitinase is essential for the growth and development of insects.Among the numerous chitinases in insects,group Ⅱ chitinase(ChtⅡ)contains the greatest number of catalytic domains and chitin-binding domains.In Lepidopterans.ChtⅡ and two other chitinases.ChtI and Chi-h,are essential for epidermal chitin hydrolysis.Although Chi-h and ChtI have been well studied,the role of ChtⅡ remains elusive.Currently,studies about ChtⅡare limited to the gene level.Down-regulated ChtⅡ gene expression by RNAi resulted in death caused by defective molting,which make ChtⅡ as a potential insecticide target.Therefore,research about ChtⅡ inhibitor can also help the development of new pesticides.GH84 O-β-N-acetylglucosaminidase is the glycoside hydrolase that catalyzes the removal of GlcNAc attached to serine/threonine residues(O-GlcNAc),which is essential for maintaining the normal O-GlcNAc level in the cell.O-GlcNAc modification is a dynamic posttranslational modification in eukaryotes and is known to occur on thousands of proteins involved in a myriad of cellular processes,including signal transduction,cell cycle,gene transcription and translation.Abnormal O-GlcNAc level is associated with many diseases,like diabetes,neurodegenerative diseases and cancer,etc.O-β-N-acetylglucosaminidase not only directly influences the physiological processes related to O-GlcNAc but also indirectly affects more signaling pathways through the interaction between O-GlcNAc and phosphorylation.In addition,the inhibitors against O-β-N-acetylglucosaminidase have potential application value during the study and treatment of diseases associated with O-GlcNAc disorders.This dissertation focused on the group Ⅱ chitinase from the agricultural pest Ostrinia furnacalis(OfChtⅡ)and O-β-N-acetylglucosaminidase from human(hOGA).The main results obtained by this work include:(1)Gene and protein sequence of OfChtⅡAccording to the highly conserved sequences based on the multiple sequence alignment of other known ChtⅡ,a 9077-nucleotide gene(GenBank ID,MF034108.1)was obtained from the cDNA of O.furnacalis adult through homologous sequence acquisition and multi-step RACE.The gene ID contains an open reading frame(157-8943 nucleotide)encoding a protein containing 2929 amino acids(GenBank ID,AS066823.1).Analyzing the domain structure of the predicted protein revealed that OfChtⅡ contains five GH18 domains and seven CBM 14-type chitin-binding modules(CBMs).The catalytic residues were mutated in three of the five GH18 domains,only the second and third GH18 domains near the C terminal are predicted to be catalytically active.(2)Hydrolysis activities and structural characteristics of OfChtⅡ truncationsFull-length OfChtⅡ is unstable and difficult to recombinant in vitro,so we constructed and expressed four OfChtⅡ truncations:OfChtⅡ-C1,OfChtⅡ-C2,OfChtⅡ-C1C2 and OfChtⅡ-B4C1.Hydrolytic activity assays showed that all the OfChtⅡ truncations exhibited low activities toward the small molecule substrate,but the activities toward polymeric substrates are highly relevant to the number of domains in the OfChtⅡ truncation.indicating that full-length ChtⅡmay have a high potential for hydrolyzing polymeric chitin.The crystal structures of two catalytically active domains of OfChtⅡ.OfChtⅡ-C1 and OfChtⅡ-C2,were obtained both in unliganded form and complexed with chitooligosaccharide substrates.OfChtⅡ-C1 and OfChtⅡ-C2 both consist of a core domain and an insertion domain.The core domain is a classic(β/α)8 barrel.OfChtⅡ-C1 and OfChtⅡ-C2 both possess a long and deep substrate-binding cleft in the surface,which exhibited structural characteristics within the substrate-binding cleft similar to those in OfChi-h and OfChtⅠ.OfChtⅡ lacks structural elements favoring substrate binding beyond the active sites.but its multiple CBMs may make up for this deficiency.(3)Small molecule inhibitors for O/Chtll and their inhibition mechanismsSeveral small molecule inhibitors of OfChtⅡ were obtained by seceening,and the inhibition mechanisms of some inhibitors were studied through enzyme-inhibitor complexes.(G1cN)8 occupied the-3 to +5 sites of the substrate-binding cleft and interacted with the enzyme mainly via stacking interactions between sugar rings and aromatic residues;Compound 2-8-S2 occupied the hydrophobic cavum between +1 and +2 site of the substrate-binding cleft,one protein could bind two inhibitors,which formed a four-layered sandwich structure with two conserved tryptophan through π-π stacking.(4)Small molecule inhibitors for hOGA and their inhibition mechanismsA series of small molecule inhibitors with new skeleton structures were designed and synthesized,including 2’ derivatives of NAG-thiazoline,NAM-thiazoline derivatives with mannose configuration,and thioglycosyl-naphthalimide hybrid molecules.The inhibition activity and selectivity between GH20 and GH84 were measured,and the inhibition mechanisms of some compounds were elaborated by structural biology and molecular docking methods.The thiazoline ring of NAM-thiazoline derivatives extends into the active pocket and stabilized by hydrogen bonds,which are similar with those found in NAG-thiazoline.For thioglycosyl-naphthalimide hybrid molecules,GH20 and GH84 enzymes showed different patterns of binding modes. |
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