Ancestral sequence reconstruction is a powerful method for inferring ancestors of modern enzymes and for studying structure–function relationships of enzymes. We have previously applied this approach to haloalkane dehalogenases (HLDs) from the subfamily HLD-II and obtained thermodynamically highly stabilized enzymes (ΔT_m up to 24 °C), showing improved catalytic properties. Here we combined crystallographic structural analysis and computational molecular dynamics simulations to gain insight into the mechanisms by which ancestral HLDs became more robust enzymes with novel catalytic properties. Reconstructed ancestors exhibited similar structure topology as their descendants with the exception of a few loop deviations. Strikingly, molecular dynamics simulations revealed restricted conformational dynamics of ancestral enzymes, which prefer a single state, in contrast to modern enzymes adopting two different conformational states. The restricted dynamics can potentially be linked to their exceptional stabilization. The study provides molecular insights into protein stabilization due to ancestral sequence reconstruction, which is becoming a widely used approach for obtaining robust protein catalysts.

祖先序列重建是推断现代酶的祖先和研究酶的结构-功能关系的有效方法。我们之前已将这种方法应用于 HLD-II 亚家族的卤代烷脱卤酶 (HLD)，并获得了热力学高度稳定的酶（Δ T _m高达 24 °C），显示出改进的催化性能。在这里，我们结合了晶体结构分析和计算分子动力学模拟，以深入了解祖先 HLD 成为具有新颖催化特性的更强大酶的机制。重建的祖先表现出与其后代相似的结构拓扑，除了一些循环偏差之外。引人注目的是，分子动力学模拟揭示了祖先酶的有限构象动力学，它们更喜欢单一状态，而现代酶则采用两种不同的构象状态。受限的动态可能与其异常的稳定性有关。该研究提供了通过祖先序列重建来实现蛋白质稳定性的分子见解，这正在成为获得稳健蛋白质催化剂的广泛使用的方法。