Hook loop dynamics engineering transcended the barrier of activity-stability trade-off and boosted the thermostability of enzymes.

The improvement of enzyme thermostability often accompanies the decreased activity due to the loss of the key regions' flexibility. As a representative structure, unlocking the potential of loop dynamics will not only provide new ideas for stabilization strategies, but also help to deepen the understanding of the relationship between enzyme structural dynamics and function. In this study, a creative "hook loop dynamics engineering" (HLoD) strategy was successfully proposed for simultaneously improving the thermostability and maintaining activity of the model enzyme, Candida Antarctica lipase B. A small and smart mutant library involving five key residues located at the "hook loop" was meticulously identified and systematically investigated and thus yielded a five-point multiple mutant M1 (L147S/T244P/S250P/T256D/N292D), demonstrating a remarkable 7.0-fold increase in thermostability at 60 °C compared to the wild-type (WT). Furthermore, the activity of M1 remained comparable to that of WT, effectively transcending the barrier of activity-stability trade-off. Molecular dynamics simulations revealed that the precise regulation of hook loop dynamics via intermolecular interactions, such as salt bridges and hydrogen bonding, curbed the excessive flexibility of the pivotal regions α5 and α10 at high temperatures, thus driving the substantial enhancement of the thermostability of M1. Refining the dynamics of the flexible region via HLoD, which transcended the barrier of activity-stability trade-off, exhibited to be a robust and potentially universal strategy for designing enzymes with outstanding thermostability and activity.