Structure And Function Study Of Two Chitinases With Antifungal Activity

In nature,there are some pathogenic fungi that can cause plant fungal diseases.The use of chemical fungicides has brought serious environmental problems.Therefore,more and more people are turning their attention to biological pesticides.The main component of the cell wall of most plant pathogenic fungi is chitin,which makes chitinase a potential antifungal agent.This article separately studied the class B chitinase AnChiB from Aspergillus niger and the family IV chitinase OfChtIV from the midgut of Corn borer.The antifungal activity was speculated and verified based on their physiological function,and the structure-function relationship was studied.The main experimental contents and results are as follows:1.Antifungal activity of AnChiB:Based on the physiological function of AnChiB,its ability to hydrolyze Aspergillus niger mycelium was determined,and 1.4 m M（GlcNAc）₂ could be generated after 8 h of hydrolysis.Subsequently,its antifungal activity was tested.The result showed that AnChiB had no effect on the growth of Phytophthora infestans whose main component of cell wall is cellulose,but had a 23%growth inhibition rate for Fusarium graminearum whose main component of cell wall is chitin.It shows that the antifungal activity of AnChiB is related to the hydrolysis of chitin in fungal cell walls.2.Structure and function of AnChiB:We compared the mycelial degradation activity of AnChiB and OfChtI,the latter being a key enzyme from the degradation process of insect cuticle,and found that the efficiency of AnChiB’s hydrolysis of mycelium is significantly higher than OfChtI,a difference of 8.7 times for the amount of（GlcNAc）₂ produced after 8 h of hydrolysis.The crystal structure of AnChiB was obtained and analysised,which is composed of a typical（β/α）₈-barrel-shaped core domain and a chitin insertion domain as a whole.And it is very similar to the crystal structure of OfChtI.The difference is that AnChiB has a LOOP structure at the non-reducing end of the substrate binding cleft,which contains Trp¹⁰⁶ and Trp¹¹⁸.We speculated that it may be related to the efficiency of mycelial hydrolysis.After the Trp¹⁰⁶ of AnChiB was mutated to Ala¹⁰⁶,the mycelial hydrolysis activity decreased by 75%,which verified the importance of the tryptophan for AnChiB’s hydrolysis of mycelium.3.Antifungal activity of OfChtIV:It was detected that OfChtIV has a 40%growth inhibition rate against F.graminearum,indicating that OfChtIV has a better antifungal activity than AnChiB.At the same time,OfChtIV was also detected to be resistant to several other corn pathogenic fungi.Since corn is often infected by fungi,this may correspond to the immune function of OfChtIV in the body of the corn borer.Subsequently,hydrolysis experiments were carried out on these kinds of fungal mycelium,and the hydrolysis efficiency was consistent with the antifungal effect,which indicated that the antifungal activity of OfChtIV also comes from the hydrolysis of the mycelium.4.Structure and function of OfChtIV:The adaptation mechanism of OfChtIV and its deglycosylation（DG）mutants under the environment of various proteases in the midgut was studied by treating the enzymes with the midgut extract.The results showed that OfChtIV was not degraded and keeped the enzyme activity;but DG-OfChtIV was degraded by more than 90%and losed 90%of the enzyme activity.It shows the protective effect of glycosylation on the enzyme under midgut conditions.Analyzing the crystal structure revealed that there are multiple protease cleavage sites on the surface of OfChtIV,further indicating the protective effect of glycosylation on the enzyme.The total number of electrostatic interactions in OfChtIV is significantly less than that of common insect chitinase,which may promote its stability under alkaline conditions in the midgut.In addition,OfChtIV is structurally more similar to plant chitinases with antifungal activity than insect chitinase.The crystals structures of the enzyme-substrate complex and the enzyme-inhibitor complex were obtained,which is helpful for a better understanding of its mode of action.