Designing ASSMD Strategy for Exploring and Engineering Extreme Thermophilic Ancestral Nitrilase for Nitriles Biocatalysis

Enzyme thermostability is vital for prolonged reactions and reusability. Extremozymes, known for their high thermal stability, have gained biocatalysis prominence. Extremozymes evolve under extreme conditions, possibly becoming extinct during the evolutionary process of adapting to the current environment. Fortunately, ancestral enzyme sequence reconstruction could deduce the ancestral enzyme sequence of existing enzymes through computer algorithms. Here, we designed an ancestral sequence-structure-molecule dynamics (ASSMD) strategy to unveil molecular insights into extinct ancestral enzymes in the evolutionary landscape. Furthermore, this approach was applied to explore the extremophilic ancestral nitrilase. In a dynamic flexibility trough, we obtained ASR135, an ancestral nitrilase capable of tolerating 90 °C. Combining evolution analysis and laboratory evolution, we achieved laboratory further evolution of the thermostability of ASR135 in this evolutionary event and obtained the mutant ASR135-M4 (S97E/S101A/N124H/H155Y), which exhibited hydrolytic activity at 100 °C. Mechanistic analysis revealed that ASR135-M4 exhibited the addition of salt bridge, hydrogen bond, and π-alkyl interaction tetrahedral cage and strengthening of the hydrophobic core inside the protein. These modifications resulted in a more robust interaction network between four secondary structures. In general, the ASSMD strategy holds potential for discovering high-performance nitrilases, particularly extremozymes. Additionally, the laboratory thermostability evolution of ASR135-M4 sheds light on enzyme-directed evolution and thermostability mechanisms.