Enzymes are essential biological catalysts that underpin vital biochemical processes through their unparalleled catalytic efficiency, constituting fundamental determinants of cellular bio-reaction (Porter et al., 2015). Beyond fundamental biological functions, enzymes have revolutionized industrial biotechnology through diverse applications: lactase in dairy processing (Movahedpour et al., 2021), proteases in pharmaceutical synthesis (Song et al., 2023), and pollutant-degrading enzymes in environmental remediation (Alcalde et al., 2006). The increasing utilization of enzymatic resources is revolutionizing synthetic pathways for high-value-added products such as pharmaceutical intermediates and fine chemicals (Naim et al., 2024). Conventional chemical processes characterized by energy-intensive conditions (e.g., high-temperature and high-pressure operations) and environmentally detrimental practices (e.g., heavy metal catalysis) are being progressively supplanted by enzymatic engineering approaches (Wohlgemuth, 2024). This paradigm shift, driven by advancements in enzyme research, is fundamentally transforming traditional industrial production models, particularly within the domains of synthetic biology and green chemistry, where biocatalytic systems demonstrate exceptional potential for sustainable manufacturing. However, natural enzymes often require optimization for industrial applications, such as enhanced enzymatic activity, stability, and environmental tolerance (Ndochinwa et al., 2024). The recent advancements in protein engineering have provided systematic solutions to address the inherent limitations of natural enzymes (Listov et al., 2024).

A comprehensive understanding of protein structures is fundamental to progress in protein engineering. Current mainstream strategies primarily utilize site-directed mutagenesis, a theoretically grounded technique, to precisely modify specific amino acid positions, thereby enhancing protein functionality (Drufva et al., 2020). Alternatively, surface charge engineering can introduce or eliminate particular amino acid residues, altering surface charge distribution and hydrophobicity, which consequently improves solubility, stability, and molecular interactions (Pedersen et al., 2019). In contrast, directed evolution adopts a demand-driven approach, frequently revealing unexpected mutational insights that expand our knowledge of structure-function relationships in proteins (Sellés Vidal et al., 2023). However, the scientific community has not yet fully elucidated the intrinsic principles governing the structure-function relationship of proteins (Ding et al., 2022; Kingsley and Lill, 2015; Rajakumara et al., 2022). This fundamental conundrum not only constitutes the core challenge in rational design strategies but also stands as the ultimate objective urgently demanding resolution in modern protein engineering. At its core, the intricate properties exhibited by protein molecules—manifested through dynamic conformational changes at the global level and response mechanisms of local secondary structures—surpass the analytical capabilities of existing theoretical frameworks.

Proteins adopt hierarchical architectures ranging from amino acid sequences (primary structure) to organized spatial arrangements—secondary (α-helices, β-sheets), tertiary, and quaternary structures—that determine biological function. These three-dimensional conformations arise from the folding of secondary structures through noncovalent interactions (Haliloglu and Bahar, 2015). While loops lack regular geometric configurations, they play an equally crucial role as connecting segments between α-helices and β-sheets in protein architecture. Notably, loops constitute 20 %–40 % of protein architecture and exhibit diverse functional roles across structural domains. Emerging research highlights specific loops as critical modulators of enzymatic activity and stability. In Triosephosphate Isomerase (TIM), a central glycolytic enzyme regulating metabolic flux, a catalytic loop undergoes hinge-like motions between open and closed conformations. This transition between substrate access (open state) and optimal catalysis (closed state) during DHAP-GAP interconversion, thereby dramatically enhancing the enzyme's catalytic efficiency (Aparicio et al., 2003). Similarly, in thermophilic bacterial PET-degrading cutinases, the coordination of Ca2+ stabilizes the open conformation of β3-α2 loop, simultaneously boosting catalytic activity and structural stability (Miyakawa et al., 2014). It is undeniable that loops can exert decisive influences on enzymatic functions and structural stability in certain cases.

While loops, distributed throughout protein structures, primarily serve as connectors between α-helices and β-sheets. Their functions, however, extend far beyond this structural role, often encompassing sophisticated regulatory and catalytic roles. For instance, in the Thermobifida fusca GH5_2 subfamily enzyme TfCel5A, three critical loops near the active site (loops 8, 3, and 7) independently manage substrate binding, intermediate stabilization, and product release (Wu et al., 2023). Some loops near active sites act as regulatory ‘lids’, controlling substrate access to the catalytic pocket. The polyether esterase SulE exemplifies this, as its lid loop participates in substrate recognition and binding while regulating conformational transitions between open and closed states (Liu et al., 2023) (Fig. 1a). Loops on ther surface of enzymes, may mediate ligand or analog binding, or modulate enzymatic activity by influencing surface electrostatic properties (Hjörleifsson et al., 2020; Jiang et al., 2012; Klein and Bachelier, 2006; Sagong et al., 2021) (Fig. 1b). In multi-subunit proteins, loops located in the interface between subunits are essential for stabilizing the protein complex (Balachandran et al., 2022) (Fig. 1c). Remarkably, distal loops, positioned far from the active site, can influence enzymatic activity through dynamic coupling and long-range interactions (Harada et al., 2014; Ouedraogo et al., 2023). Beyond these functional categories, unique loop configurations exist. For example, in Cytochrome P450 enzymes, the BC loop (connecting helices B and C) acts as a structural hinge, forming a solvent-accessible cavity for substrate binding (Takahashi et al., 2022) (Fig. 1d). Another notable example is the Ω-loop, a defining feature of β-lactamases, which plays a central role in determining antibiotic side-chain specificity (Nukaga et al., 2004). Studies have shown that modifications in the Ω-loop, including mutations, insertions, and deletions, are primary mechanisms underlying bacterial antibiotic resistance (Banerjee et al., 1998; Endimiani et al., 2010). It is noteworthy that the inherent conformational plasticity of loops suggests enzymatic functions may not be singular, as their broad ensemble of accessible conformations could enable these structural motifs to adopt multifunctional roles (Liang et al., 2018).

In summary, although loop regions serve as dynamic structural elements connecting rigid secondary structures and are extensively involved in the core functionalities of enzymes, systematic investigations into these functional components remain notably deficient. Current research predominantly focuses on elucidating the global structure-function relationships of enzymes, primarily examining molecular design strategies and underlying principles from a holistic perspective. This approach has resulted in two critical research gaps: (i) insufficient elucidation of structure-function correlations specific to loop architectures, and (ii) a lack of comprehensive summarization regarding characterization methodologies and systematic engineering frameworks for loop modification. Given the functional significance of loops in enzyme structures, this review outlines their fundamental roles and elucidates representative molecular mechanisms. Recognizing methodological limitations in loop research, we have organized experimental and computational approaches to facilitate investigations into loop functions. However, the structural flexibility of loops presents challenges in developing a comprehensive theoretical framework for their engineering. To address these challenges, we have curated prominent examples of successful loop engineering from recent studies, highlighting diverse strategies for their modification and application.