Loop engineering in enzymes from structure to function: Mechanisms, methodologies, and engineering strategies

Enzymes play indispensable roles not only in biological processes but also in industrial applications such as chemical manufacturing, food processing, and medicine. The loop, contrasting with the rigid conformations of α-helices and β-sheets, significantly influences the catalytic performance and environmental adaptability due to its structural and conformational diversity. Given this flexibility, loop engineering emerges as a key strategy to modulate enzyme properties. This review aims to provide a systematic overview of loop functions and their engineering. Specifically, it (1) summarizes the physiological functions associated with key loops, (2) elucidates the underlying molecular mechanisms, (3) outlines current experimental and computational methodologies for loop investigation, and (4) compiles state-of-the-art strategies for loop engineering. The novelty of this review lies in its comprehensive synthesis of recent advances and practical applications specifically focused on loop research, an area crucial for understanding protein structure-function relationships but often less emphasized than other secondary structures. Therefore, a systematic introduction to loops through this review is of great significance for understanding the structure-function relationships of proteins and enhancing enzyme performance.