Crystal Structure And Inhibitor Design Of A Chitinase From The Insect Ostrinia Furnacalis

Glycoside hydrolase family18（GH18） chitinases （EC3.2.1.14） catalyze the random hydrolysis of N-acetyl-β-D-glucosaminide （1â†'4）-β-linkages in chitin and chitodextrins. They are widely distributed in many organisms including bacteria, fungi, nematodes, insects, plants and mammals, and play vital roles in nutrition, pathogenicity, moulting, and immunity and defence. Understanding the structure-function relationship of these enzymes is essential for disease control, plant protection and drug design.Insects possess a greater number of chitinases than any other organisms; in fact, an insect may possess as many as eight groups of genes encoding GH18chitinases. Among the eight groups of insect chitinases, group I chitinases have been shown to function in a chitin-degradation process that is closely associated with insect moulting, and thus plays an essential role in insect development. Unfortunately, there is no structural information available for insect chitinases. This dissertation focused on the group I chitinase OfChtI from the destructive pest Ostrinia furnacalis. The main conclusions by this work include:1) OfChtl possesses unique structural characteristicsRecombinant full-lengthed OfChtl is not stable. Thus the catalytic domain of OfChtI （OfChtI-CAD） was expressed and crystallized. The structure of OfChtI-CAD （PDB entry3w4r） was solved at a resolution of1.7A.The crystal structue indicates OfChtI-CAD folds into two distinct domains:a core domain and an insertion domain. The core domain is a classical （p/a）8-barrel fold with the catalytic motif ’DxDxE’（Asp144-Glu148） located in the loop between the strand β4and the helix a4. And the insertion domain forms one of the walls of the active cleft.A long substrate-binding cleft with both ends open was observed on the surface of OfChtI-CAD. This cleft is composed of9lined-up aromatic residues deduced for binding with sugar residues of substrates. Compared with other structure-known chitinases,OfChtI-CAD possesses a unique hydrophobic plane characterized by four solvent-exposed aromatic residues, Phe159, Phe194, Trp241and Tyr290, adjacent to the end of the substrate-binding cleft. Single-site mutantions （F159A, F194A, W241A and Y290A）, double-site mutantions （F194A/W241A, F159A/Y290A） and four-site mutantions （F159A/F194A/W241A/Y290A） partially lost chitin-binding capacity when compared with the wild-type. The most significant decrease in chitin-binding capacity was observed for the four-site mutation, which exhibits40%of binding ability of wild-type OfChtl-CAD. These results suggest that the four residues forming a unique hydrophobic plane are involved in chitin binding. 2) OfChtl-CAD is an endo-acting enzyme towards chitooligosaccharidesTo investigate the hydrolysis property of OfChtI-CAD, we analyzed the binding of chitooligosaccharides to OfChtI-CAD. The results indicated that the binding of （GlcNAc）3-6to OfChtI-CAD were exothermic （△H<0） and entropically driven with an enthalpic contribution （△H<0,|-Tâ–³S|>|â–³H）. This evidence suggests that substrate binding is accompanied by desolvation and conformational changes of both the protein and its ligands. An increase in the polymerization degree of GlcNAc （from （GlcNAc）3to（GlcNAc）6）, results in an average free-energy gain of approximately-1.0kcal mol-1per GlcNAc residue, demonstrating that OfChtI possesses at least six substrate-binding subsites.To investigate the catalytic property, the crystal of OfChtI-CAD was soaked with （GlcNAc）6. The complex structure （PDB entry3wll） was then solved at a resolution of1.77A. Structure comparison indicated that the complex structure of OfChtI-CAD with （GlcNAc）6is well-superimposed with the unliganded enzyme, suggesting the binding of substrate does not introduce major changes in eithor the catalytic active site or the overall structure. Two sugars were found in the complexed OfChtI-CAD-（GlcNAc）6. One was （GlcNAc）3occupying the subsites-1,-2and-3, and the other （GlcNAc）2occupied the subsites+1and+2. It was worth noting that the-1GlcNAc residue was in an energetically unfavoured’boat’ conformation. This conformation was likely to be a state that occurred just before the completion of catalysis, in which the product had left and the catalysis-associated Asp146residue was still interacting with the-1sugar. Because our previous studies evidenced that OfChtI catalyzed the hydrolysis from the nonreducing ends of substrates, the （GlcNAc）3with a reducing sugar at the subsite-1suggested that （GlcNAc）3could be a hydrolysis product of （GlcNAc）6. Thus, OfChtI-CAD was concluded to possess endo-chitinase activity.3) Fully-deacetylated chitooligosaccharides acting as OfChtI inhibitorsGH18chitinases catalyze hydrolysis through a substrate-assisted mechanism in which the C2N-acetyl group of the-1sugar of a substrate is the nucleophile that attacks the C1carbon. Binding of GlcNAc in the-1subsite requires considerable distortion and is energetically unfavorable. Therefore, we hypothesized that if the-1sugar was deacetylated, it might be energetically favorable to bind a GlcN in the-1subsite. We first reported that a series of fully deacetylated chitooligosaccharides （GlcN）2-7could act as inhibitors against GH18chitinases with IC50values at the micromolar to millimolar levels.The in vivo activities were also promising. The injection of mixed （GlcN）2-7into the fifth instar larvae of the insect Ostrinia furnacalis resulted in85%of the larvae being arrested at the larval stage and death after10days. As OfChtI functions in the chitin-degradation process during insect moulting, the results also suggested that （GlcN）2₇might inhibit OfChtI in vivo.4) Inhibitory mechanism of fully deacetylated chitooligosaccharides To investigate the inhibitory mechanism of fully deacetylated chitooligosaccharides against OfChtI, the thermodynamics of the binding of （GlcN）2-7to OfChtI was determined by using isothermal titration calorimetry （ITC）. The binding of all of the inhibitors examined was exothermic （△H<0） and enthalpically driven （|â–³H|>|-TAS|）. These data suggested that inhibitor binding was accompanied by hydrogen bonding and/or electrostatic-electrostatic interaction. The similar Kd values for （GlcN）5,（GlcN）6and （GlcN）7binding to OfChtI indicated that the additional GlcN residues of （GlcN）6,7could not contribute to the binding affinity.To structurally investigate the molecular mechanism of inhibition, we crystallized and obtained complex structures of OfChtI-CAD with （GlcN）5（PDB entry3wqv） and （GlcN）6（PDB entry3wqw）, respectively. Both of the complex structures were solved at a resolution of2.0A. In OfChtI-CAD-（GlcN）5and OfChtI-CAD-（GlcN）6, the five sugars of both ligands occupied the same subsites, which were-1,-2,-3,-4and-5subsites. A structural comparison of the inhibitor-enzyme complex, OfChtI-CAD-（GlcN）5, with the substrate-enzyme complex （OfChtI-CAD-（GlcNAc）2/3） revealed that the binding modes of ligands in the-1and-2subsites were different. To investigate the roles of these two subsites in inhibitor binding, the mutants E148Q and F309A were constructed. Thermodynamics analysis showed that the binding of the inhibitor （GlcN）5to the mutant E148Q exhibited19-fold lower affinity if compared with the wild type. Enzymatic analysis indicated that E148Q lost the enzymatic activity, and the mutation of the Phe309to Ala309resulted in a4-fold inhibitory activity decrease. Therefore, we concluded that the-1and-2subsites were crucial for GlcN oligomers inhibiting the activity of OfChtI.In summary, this work is in favor of understanding the relationship between multiple physiological functions and accordingly various structures of chitinases, and is of great significance to the development of new technology for insect-mediated disease therapy and agricultural pest control.