Removal ofN-linked glycans in cellobiohydrolase Cel7A fromTrichoderma reeseireveals higher activity and binding affinity on crystalline cellulose

Background Cellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. For many years,Trichoderma reeseiCel7A (TrCel7A) has served as a model to understand structure-function relationships of processive cellobiohydrolases. The architecture ofTrCel7A includes anN-glycosylated catalytic domain, which is connected to a carbohydrate-binding module through a flexible,O-glycosylated linker. Depending on the fungal expression host, glycosylation can vary not only in glycoforms, but also in site occupancy, leading to a complex pattern of glycans, which can affect the enzyme's stability and kinetics. Results Two expression hosts,Aspergillus oryzaeandTrichoderma reesei, were utilized to successfully express wild-typesTrCel7A (WT(Ao)and WTTr) and the tripleN-glycosylation site deficient mutantsTrCel7A N45Q, N270Q, N384Q (Delta N-glyc(Ao)and Delta N-glyc(Tr)). Also, we expressed singleN-glycosylation site deficient mutantsTrCel7A (N45Q(Ao), N270Q(Ao), N384Q(Ao)). TheTrCel7A enzymes were studied by steady-state kinetics under both substrate- and enzyme-saturating conditions using different cellulosic substrates. The Michaelis constant (K-M) was consistently found to be lowered for the variants with reducedN-glycosylation content, and for the triple deficient mutants, it was less than half of the WTs' value on some substrates. The ability of the enzyme to combine productively with sites on the cellulose surface followed a similar pattern on all tested substrates. Thus, site density (number of sites per gram cellulose) was 30-60% higher for the single deficient variants compared to the WT, and about twofold larger for the triple deficient enzyme. Molecular dynamic simulation of theN-glycan mutantsTrCel7A revealed higher number of contacts between CD and cellulose crystal upon removal of glycans at position N45 and N384. Conclusions The kinetic changes ofTrCel7A imposed by removal ofN-linked glycans reflected modifications of substrate accessibility. The presence ofN-glycans with extended structures increasedK(M)and decreased attack site density ofTrCel7A likely due to steric hindrance effect and distance between the enzyme and the cellulose surface, preventing the enzyme from achieving optimal conformation. This knowledge could be applied to modify enzyme glycosylation to engineer enzyme with higher activity on the insoluble substrates.