| OsXIP（Oryza sativa xylanase inhibitor protein）is a recently discovered XIP-type xylanase inhibitor（XI）found in rice.It serves as a defense protein against xylanases secreted by fungal pathogens,preventing the degradation of the cell wall.At present,therewas little information available about inhibition type,structural basis and molecular mechanism between OsXIP and family GH10 xylanase.In this study,the genes encoding family GH10 xylanases,afxyla10 and xyn-lxy＿cd from Hu sheep rumen contents werecloned and successfully recombinant expressed in Pichia pastoris X33.The inhibition interactions between OsXIP and the two xylanases wereinvestigated.1.The family GH10 xylanase genes,A.fumigatus Xylanase A afxyla10 and xyn-lxy＿cd from Hu sheep rumen contents werecloned and recombinantly expressed in P.pastoris X33,positive expression strains of PPafxyla10-2 and PPxyn-lxy＿cd-8werescreened.The open reading frame（ORF）of afxyla10（Gen Bank:MT682384.1）was 1080 bp,encoding the 359 amino acids residues with a theoretical molecular weight of 40.0 k Da.The recombinant xylanase reAfXylA10 was about 52.0 k Da with activity of 18.63 U/m L;the optimum temperatureand p H of reAfXylA10 was53°C and 7.0,respectively.Mn2+remarkably activated the catalytic activity.The xyn-lxy＿cd（Genbank:KP723541.1）was 1182 bp,which encodes 394 amino acids（50-443 aa）with a predicted molecular mass of 44.4 k Da.The recombinant xylanase reXYN-LXY＿CD was secreted in the culturewith activity of 58.08 U/m L.The molecular weight of reXYN-LXY＿CD was approximately 56.0 k Da.The optimum temperatureand p H of reXYN-LXY＿CD was 53°C and 7.0,respectively.Mn2+and Co2+remarkably activated its catalytic activity.2.Exploring the inhibition interaction of reOsXIP on reAfXylA10 and reXYN-LXY＿CD.rePOsXIP competitively inhibited the catalytic activity of reAfXylA10 and reXYN-LXY＿CD with inhibition constant（Ki）of 177.94 n M and237.27 n M,respectively.Effect of rePOsXIP on hydrolytic performance of reXYN-LXY＿CD was analyzed by HPLC.The concentration of hydrolysis products from beechwood xylan by reAfXylA10 and reXYN-LXY＿CD decreased in the presence of rePOsXIP,whereas types of hydrolysates werenot changed.Fluorescence spectroscopy analysis showed the fluorescence intensity of two xylanases decreased,suggesting the fluorescence of reAfXylA10 and reXYN-LXY＿CD werestatically quenched by rePOsXIP.3.Molecular dynamics（MD）simulations and non-covalent interaction analysis wereused to elucidate enzyme-inhibitor interaction.The systems of AfXylA10-OsXIP and XYN-LXY＿CD-OsXIP wereconstructed by homology modeling and molecular docking,and then 100-ns MD simulations wereconducted subsequently.Results of MD simulations showed the inhibition interaction between xylanases and OsXIP weredominated by non-covalent interactions,residue K239 on theα-7helix of OsXIP specifically interacts with the acid-base catalytic residue E140AfXylA10 and E197XYN-LXYCD through hydrogen bond and vd W interaction.Besides,residue pairs T104AfXylA10/V99AfXylA10-Q5OsXIP,R256AfXylA10-E235OsXIP,D155AfXylA10-Y243OsXIP,D145AfXylA10-R194OsXIPand E229OsXIP-T331XYN-LXY＿CD/F332XYN-LXY＿CD,R272OsXIP-H222XYN-LXY＿CD,N6OsXIP-N216 XYN-LXY＿CD,K247OsXIP-D201XYN-LXY＿CD formed on the upper of the TIM barrel around the catalytic cleft werealso played a strong role.4.MD simulations and site-directed mutation wereperformed based on AfXylA10-OsXIP system.Sites V99,D145,D155,and E309/Q311 of AfXylA10wereselected for Ala mutation.Inhibition system of OsXIP and the 9 mutants（V99A,D145A,D155A,V99A/D145A,V99A/D155A,D145A/D155A,V99A/D145A/D155A,E309A/Q311A,and V99A/D145A/E309A/Q311A）wereconstructed.Results of MM-PBSA calculation and hydrogen bond analysis showed that residues involved in the inhibition of OsXIP-V99A mutant weresignificantly reduced.Therewas no significant change in the interaction of OsXIP on mutants D145A,D155A,V99A/D145A,and V99A/D155A.The inhibition interaction on the left loop and catalytic pocket of OsXIP and mutants D145A/D155A,V99A/D145A/D155A,E309A/Q311A,and V99A/D145A/E309A/Q311A werestrengthened.The nine mutants wereconstructed by over-lap PCR and successfully expressed in P.pastoris X33.The experimental results show that the inhibition activities of rePOsXIP on mutant enzymes V99A and V99A/D145A decreased significantly.The inhibition activitis of rePOsXIP on D155A and V99A/D155A decreased,but the difference was not significant.The inhibition activities of rePOsXIP on the rest five mutant enzymes（D145A,D145A/D155A,V99A/D145A/D155A,E309A/Q311A,and V99A/D145A/E309A/Q311A）weresignificantly enhanced.The results of experimental inhibition of rePOsXIP on V99A,D145A/D155A,V99A/D145A/D155A,E309A/Q311A,and V99A/D145A/E309A/Q311A wereconsistent with MD simulation results.In conclusion,the two GH10 xylanases wererecombinantly expressed in P.pastoris and characterized.rePOsXIP competitively inhibited both reAfXylA10 and reXYN-LXY＿CD.MD simulations,non-covalent interaction analysis and site-directed mutation wereused to elucidate the mechanism of the enzyme-inhibitor interaction.This study might provide the molecular basis for designing family GH10mutants resistant to OsXIP while maintaining high catalytic activity. |
