Engineering catalytically promiscuous enzymes to serve new functions

Catalytic promiscuity in enzymes refers to their ability to catalyze multiple chemically distinct reactions in addition to their native activity. The increasing discovery of additional enzymes exhibiting catalytic promiscuity has underscored the significance of this trait in nature. The catalytic promiscuity of enzymes offers new avenues for functional redesign. Through protein engineering, existing enzymes can be modified to expand their natural catalytic boundaries. Furthermore, de novo designed artificial enzymes can achieve novel enzymatic reactions, broadening the scope of enzyme-catalyzed applications. Given that catalytic promiscuity plays a fundamental role in enzyme evolution, comprehensive research on its origins and influencing factors is essential. In this review, we comprehensively examine the factors influencing catalytic promiscuity, including variations in substrate binding modes in pre-reaction states, the instability of key high-energy intermediates, and the roles of critical residues in catalytic mechanisms. Moreover, altering the enzyme's catalytic environment can also induce novel types of catalytic reactions, such as light-induced promiscuous reactions catalyzed by cofactor-dependent oxidoreductase enzymes. Additionally, we summarize the current protein engineering technologies and strategies aimed at enhancing the activity and stereoselectivity of target enzymes to meet industrial requirements.