Cellobiohydrolase from glycoside hydrolase family 7 is a major component of commercial enzymatic mixtures for lignocellulosic biomass degradation. For many years, Trichoderma reesei Cel7A (TrCel7A) has served as a model to understand structure–function relationships of processive cellobiohydrolases. The architecture of TrCel7A includes an N-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. Two expression hosts, Aspergillus oryzae and Trichoderma reesei, were utilized to successfully express wild-types TrCel7A (WTAo and WTTr) and the triple N-glycosylation site deficient mutants TrCel7A N45Q, N270Q, N384Q (ΔN-glycAo and ΔN-glycTr). Also, we expressed single N-glycosylation site deficient mutants TrCel7A (N45QAo, N270QAo, N384QAo). The TrCel7A enzymes were studied by steady-state kinetics under both substrate- and enzyme-saturating conditions using different cellulosic substrates. The Michaelis constant (KM) was consistently found to be lowered for the variants with reduced N-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 the N-glycan mutants TrCel7A revealed higher number of contacts between CD and cellulose crystal upon removal of glycans at position N45 and N384. The kinetic changes of TrCel7A imposed by removal of N-linked glycans reflected modifications of substrate accessibility. The presence of N-glycans with extended structures increased KM and decreased attack site density of TrCel7A 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.

中文翻译：

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从里氏木霉中去除纤维二糖水解酶 Cel7A 中的 N 连接聚糖揭示了对结晶纤维素的更高活性和结合亲和力。

来自糖苷水解酶家族 7 的纤维二糖水解酶是用于木质纤维素生物质降解的商业酶混合物的主要成分。多年来，里氏木霉 Cel7A (TrCel7A) 一直作为模型来了解进行性纤维二糖水解酶的结构-功能关系。TrCel7A 的架构包括一个 N-糖基化催化结构域，该结构域通过一个灵活的 O-糖基化接头连接到碳水化合物结合模块。根据真菌表达宿主的不同，糖基化不仅在糖型上有所不同，而且在位点占据上也有所不同，从而导致聚糖的复杂模式，这会影响酶的稳定性和动力学。两种表达宿主，米曲霉和里氏木霉，用于成功表达野生型TrCel7A（WTTao和WTTr）和三重N-糖基化位点缺陷突变体TrCel7A N45Q，N270Q，N384Q（ΔN-glycAo和ΔN-glycTr）。此外，我们表达了单个 N-糖基化位点缺陷突变体 TrCel7A (N45QAo、N270QAo、N384QAo)。使用不同的纤维素底物在底物和酶饱和条件下通过稳态动力学研究 TrCel7A 酶。一致发现，对于 N-糖基化含量降低的变体，米氏常数 (KM) 会降低，对于三重缺陷突变体，它不到某些底物上 WT 值的一半。酶与纤维素表面位点有效结合的能力在所有测试的底物上都遵循类似的模式。因此，与 WT 相比，单一缺陷变体的位点密度（每克纤维素的位点数）高 30-60%，而三重缺陷酶则高出约两倍。N-聚糖突变体 TrCel7A 的分子动力学模拟显示，在去除 N45 和 N384 位置的聚糖后，CD 和纤维素晶体之间的接触数量增加。通过去除 N-连接聚糖而引起的 TrCel7A 的动力学变化反映了底物可及性的改变。具有扩展结构的 N-聚糖的存在增加了 TrCel7A 的 KM 并降低了攻击位点密度，这可能是由于空间位阻效应和酶与纤维素表面之间的距离，从而阻止了酶获得最佳构象。