期刊名称：Computational and Structural Biotechnology Journal
印刷版ISSN：2001-0370
出版年度：2020
卷号：18
页码：1497-1508
DOI：10.1016/j.csbj.2020.06.021
出版社：Computational and Structural Biotechnology Journal
摘要：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 T 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.
