Elucidating the catalytic principles of enzymes and constructing efficient biocatalytic systems have long been central challenges in enzyme engineering. Although various strategies such as directed evolution, rational design, and artificial intelligence have advanced rapidly, accurately predicting the effects of mutations on enzyme activity and stability remains a major bottleneck in the field.

A new study, led by Tao Tu’s group from the team of Bin Yao at the Institute of Animal Sciences of Chinese Academy of Agricultural Sciences (CAAS) has proposed and validated a novel ESTEEM (Evolutionary-Structural Tuning of Enzyme Efficiency Modules) strategy, published as a cover article in ACS Catalysis.

Using cytochrome P450 monooxygenase as a model system, the researchers integrated evolutionary conservation information with three-dimensional structural analysis to establish an evolution–structure coupling design framework, providing a new paradigm for rational enzyme engineering. Multiscale computational analyses revealed the intrinsic relationship among tunnel mutations, conformational dynamics, and catalytic efficiency, highlighting the crucial role of enzyme tunnels in evolutionary and functional optimization. The proposed ESTEEM strategy offers a generally applicable approach for the rational design of enzymes with deeply buried active sites, paving a new way for the construction of high-performance biocatalytic systems.

The study focused on daidzein 3′-hydroxylase P450 (CYP107H_B.am) as the target enzyme and systematically optimized key structural regions based on the ESTEEM framework. By integrating evolutionary information with structural features, the team developed a standardized enzyme design workflow and identified several critical “gating” residues within the substrate tunnel and active pocket that determine catalytic efficiency. Mutations in the tunnel region markedly improved substrate transport and product release, achieving a synergistic enhancement of catalytic activity and thermal stability. Further structural and dynamic analyses revealed a tight coupling between tunnel optimization and catalytic enhancement, characterized by a structural regulation mode described as “rigidifying flexible scaffolds while flexibilizing rigid ones.” This strategy not only enhanced the catalytic efficiency of P450 enzymes but also broadened their substrate adaptability toward multiple flavonoid compounds.

To verify the general applicability of the ESTEEM strategy, the researchers extended it to multiple P450 homologous systems, all of which exhibited improved catalytic activity. Subsequent analyses showed that the magnitude of activity enhancement correlated closely with the tunnel’s geometric configuration and structural tunability. These findings indicate that rational enzyme design should incorporate comprehensive evaluation of tunnel parameters—such as length, radius, and curvature—to achieve greater predictive accuracy and engineering success.

In summary, by integrating evolutionary information with structural features, the researchers established an efficient ESTEEM-based enzyme design strategy and successfully achieved a systematic enhancement of P450 catalytic performance. This study not only elucidates the critical role of “evolutionary gating” in enzyme catalytic regulation but also provides a new rational design pathway for enzymes with buried active sites, offering important theoretical and technological support for the construction of efficient biocatalysts and the green biosynthesis of high value-added functional feed ingredients.

This work was supported by the National Natural Science Foundation of China and the China Agriculture Research System of Ministry of Finance and Ministry of Agriculture and Rural Affairs programs.

Link：https://doi.org/10.1021/acscatal.5c03170