Babkova Petra, Dunajova Zuzana, Chaloupkova Radka, Damborsky Jiri, Bednar David, Marek Martin
Loschmidt Laboratories, Department of Experimental Biology and RECETOX, Faculty of Science, Masaryk University, Kamenice 5, Bld. A13, 625 00 Brno, Czech Republic.
International Clinical Research Center, St. Anne's University Hospital Brno, Pekarska 53, 656 91 Brno, Czech Republic.
Comput Struct Biotechnol J. 2020 Jun 19;18:1497-1508. doi: 10.1016/j.csbj.2020.06.021. eCollection 2020.
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 (Δ 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以及研究酶的结构-功能关系的一种强大方法。我们之前已将此方法应用于HLD-II亚家族的卤代烷脱卤酶（HLD），并获得了热力学上高度稳定的酶（Δ高达24°C），其催化特性有所改善。在此，我们结合晶体学结构分析和计算分子动力学模拟，以深入了解祖先HLD如何成为具有新催化特性的更稳定酶的机制。除了一些环的偏差外，重建的祖先与其后代表现出相似的结构拓扑。引人注目的是，分子动力学模拟揭示了祖先酶受限的构象动力学，与采用两种不同构象状态的现代酶相反，祖先酶更喜欢单一状态。这种受限的动力学可能与其异常的稳定性有关。该研究为因祖先序列重建导致的蛋白质稳定化提供了分子层面的见解，而祖先序列重建正成为获得稳定蛋白质催化剂的一种广泛使用的方法。
