Glycosidases are phylogenetically widely distributed enzymes that are crucial for the cleavage of glycosidic bonds. Here, we present the exceptional properties of a putative ancestor of bacterial and eukaryotic family-1 glycosidases. The ancestral protein shares the TIM-barrel fold with its modern descendants but displays large regions with greatly enhanced conformational flexibility. Yet, the barrel core remains comparatively rigid and the ancestral glycosidase activity is stable, with an optimum temperature within the experimental range for thermophilic family-1 glycosidases. None of the about 5500 reported crystallographic structures of about 1400 modern glycosidases show a bound porphyrin. Remarkably, the ancestral glycosidase binds heme tightly and stoichiometrically at a well-defined buried site. Heme binding rigidifies this TIM-barrel and allosterically enhances catalysis. Our work demonstrates the capability of ancestral protein reconstructions to reveal valuable but unexpected biomolecular features when sampling distant sequence space. The potential of the ancestral glycosidase as a scaffold for custom catalysis and biosensor engineering is discussed.

中文翻译：

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新型血红素结合可在古老的TIM-桶糖苷酶中进行变构调节

糖苷酶是系统发生广泛分布的酶，对于糖苷键的裂解至关重要。在这里，我们介绍细菌和真核家庭1糖苷酶的推定祖先的特殊属性。祖先蛋白与其现代后代共享TIM-桶状折叠，但显示出具有大大增强的构象灵活性的大区域。然而，桶状核保持相对刚性，祖先糖苷酶活性稳定，最佳温度在嗜热家族1糖苷酶的实验范围内。报道的约1400种现代糖苷酶的约5500种晶体结构均未显示结合的卟啉。值得注意的是，祖先糖苷酶在定义明确的掩埋位点紧密地和化学计量地结合血红素。血红素结合使该TIM桶硬化并变构增强催化作用。我们的工作证明了对远距离序列空间进行采样时，祖先蛋白重建能够揭示有价值但出乎意料的生物分子特征的能力。讨论了祖先糖苷酶作为定制催化和生物传感器工程支架的潜力。