Abstract The flexible loops or domains have been widely regarded to be involved in the catalytic modulation, substrate recognition, or thermal stability of enzymes. However, in AGE superfamily, the relationship of flexible loop and the tryptophan residue with catalytic direction and substrate specificity was still a mystery. In order to uncover this, the specific structures of cellobiose 2-epimerase (CE)-flexible loop and Trp372, were grafted into the mannose isomerase (MI) to generate a new recombinant protein named CMW. As a result, CMW showed no isomerization activity to monosaccharide-mannose but displayed epimerization and isomerization activity toward disaccharide, indicating that the CMW got close to CE but stepped far away from MI. These results demonstrated that the flexible loop and Trp372 were two key factors in determining the catalytic direction and substrate specificity. Combining the computational calculation and the structural analysis, it was speculated that: the (α/α)6-barrel of AGE superfamily only participated in the proton transfer process but didn’t guide the catalytic direction; in contrast, the epimerization activity was proposed to be determined by the tryptophan and the flexible loop and the isomerization was highly related to the residue composition and the shape change in the flexible loop. In addition, the substrate specificity for monosaccharide or disaccharide were suggested to be determined by the CsCE_Trp308 and Trp372.

中文翻译：

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深入了解 Trp372 和柔性环在指导 AGE 超家族酶的催化方向和底物特异性方面的重要作用

摘要 柔性环或结构域被广泛认为与酶的催化调节、底物识别或热稳定性有关。然而，在AGE超家族中，柔性环和色氨酸残基与催化方向和底物特异性的关系仍然是个谜。为了揭示这一点，将纤维二糖 2-差向异构酶 (CE)-柔性环和 Trp372 的特定结构嫁接到甘露糖异构酶 (MI) 中以生成一种名为 CMW 的新重组蛋白。结果表明，CMW对单糖-甘露糖没有异构化活性，但对二糖表现出差向异构化和异构化活性，表明CMW接近CE但远离MI。这些结果表明，柔性环和 Trp372 是决定催化方向和底物特异性的两个关键因素。结合计算计算和结构分析推测：AGE超家族的(α/α)6-barrel只参与质子转移过程，不指导催化方向；相比之下，差向异构化活性被提出由色氨酸和柔性环决定，异构化与柔性环中的残基组成和形状变化高度相关。此外，单糖或双糖的底物特异性建议由 CsCE_Trp308 和 Trp372 确定。AGE超家族的(α/α)6-barrel只参与质子转移过程，不指导催化方向；相比之下，差向异构化活性被提出由色氨酸和柔性环决定，异构化与柔性环中的残基组成和形状变化高度相关。此外，单糖或双糖的底物特异性建议由 CsCE_Trp308 和 Trp372 确定。AGE超家族的(α/α)6-barrel只参与质子转移过程，不指导催化方向；相比之下，差向异构化活性被提出由色氨酸和柔性环决定，异构化与柔性环中的残基组成和形状变化高度相关。此外，单糖或双糖的底物特异性建议由 CsCE_Trp308 和 Trp372 确定。