Abstract：
Dye-decolorizing peroxidases (DyPs) are recently identified microbial enzymes that have been used in several Biotechnology applications from wastewater treatment to lignin valorization. However, their properties and mechanism of action still have many open questions. Their heme-containing active site is buried by three conserved flexible loops with a putative role in modulating substrate access and enzyme catalysis. Here, we investigated the role of a conserved glutamate residue in stabilizing interactions in loop 2 of A-type DyPs. First, we did site saturation mutagenesis of this residue, replacing it with all possible amino acids in bacterial DyPs from Bacillus subtilis (BsDyP) and from Kitasatospora aureofaciens (KaDyP1), the latter being characterized here for the first time. We screened the resulting libraries of variants for activity towards ABTS and identified variants with increased catalytic efficiency. The selected variants were purified and characterized for activity and stability. We furthermore used Molecular Dynamics simulations to rationalize the increased catalytic efficiency and found that the main reason is the electron channeling becoming easier from surface-exposed tryptophans. Based on our findings, we also propose that this glutamate could work as a pH switch in the wild-type enzyme, preventing intracellular damage.
摘要：
染料脱色过氧化物酶（DyP）是最近鉴定的微生物酶，已用于从废水处理到木质素的几种生物技术应用中。然而,它们的性质和作用机制仍有许多悬而未决的问题。它们的含血红素的活性位点被三个保守的柔性环掩埋，在调节底物进入和酶催化中具有推定的作用。这里,我们研究了保守的谷氨酸残基在稳定A型DyP的第2环中的相互作用中的作用。首先,我们对这个残基进行了位点饱和诱变，用来自枯草芽孢杆菌（BsDyP）和来自金黄北孢菌（KaDyP1）的细菌DyP中的所有可能的氨基酸代替它，后者在这里第一次被定性。我们针对对ABTS的活性筛选了所得变体文库,并鉴定了具有提高的催化效率的变体。纯化所选择的变体并表征活性和稳定性。我们还使用分子动力学模拟来合理化增加的催化效率，并发现主要原因是电子通道变得更容易从表面暴露的色氨酸。根据我们的发现,我们还提出这种谷氨酸可以作为野生型酶的pH开关，防止细胞内损伤。
