New insights into exploring new functional enzymes through the enzyme promiscuity.

Enzyme promiscuity, defined as the ability of enzymes to catalyze reactions beyond their primary physiological functions, has emerged as a pivotal concept in modern enzyme engineering. This review provides a comprehensive exploration of enzyme promiscuity and its implications for the discovery and development of novel functional enzymes. Through targeted strategies such as (semi-)rational design, directed evolution, and de novo design, enzyme promiscuity has been harnessed to broaden substrate scopes, enhance catalytic efficiencies, and adapt enzymes to diverse reaction conditions. These modifications often involve subtle alterations to the active site, which impact catalytic mechanisms and open new pathways for the synthesis and degradation of complex organic compounds. Striking a balance between maintaining native activity and enhancing promiscuous functions remains a significant challenge in enzyme engineering. Nevertheless, advances in structural biology and computational modeling offer promising strategies to overcome these obstacles. By elucidating the mechanistic basis of enzyme promiscuity, this review aims to deepen our understanding of this phenomenon. It underscores the necessity of further investigating the mechanisms underlying promiscuous enzymatic activity and highlights the importance of leveraging promiscuous enzymes to address industrial application demands and drive the development of next-generation biocatalysts.