Global Dynamic Design (GDD) Empowers Cytochrome P450s for Improved Catalytic Performance
Protein engineering is a powerful tool to enhance the catalytic performance of enzymes. State-of-the-art methodologies usually focus on identifying positions in localized regions, such as the binding pockets and substrate access tunnels. Here, we presented a global dynamic design strategy to evolve cytochrome P450s for improved catalytic performance by identifying distant residues. Three potential positions (D164, A195, and E405) were identified and subjected to site saturation mutagenesis (SSM), resulting in the beneficial variant 8G8/D164N/A195P, which exhibited 7- and 148-fold improved kcat values compared to variants 8G8 and WT, respectively. Dynamic tunnel and conformational dynamic analyses revealed that substitutions distant from substrate access tunnels increased substrate translocation through the substrate access tunnels of CYP116B3, improving the catalytic performance. General applicability was demonstrated by transferring the global dynamic design strategy to an additional P450 enzyme, namely P450 BM3. The proposed global design strategy advances state-of-the-art P450 engineering by improving the catalytic performance of P450 by identifying distant correlated positions that enhance substrate translocation through access tunnels.
Authors
- Mehdi D. Davari (ORCID: https://orcid.org/0000-0003-0089-7156)
- Chenba Zhu (ORCID: https://orcid.org/0009-0008-0709-9628)
- Ulrich Schwaneberg (ORCID: https://orcid.org/0000-0003-4026-701X)
- Binbin Nian (ORCID: https://orcid.org/0000-0001-7736-0889)
- Haijun Xu (ORCID: https://orcid.org/0000-0002-8721-3310)
- Yu Ji (ORCID: https://orcid.org/0000-0002-9609-5395)
- Shuaiqi Meng (ORCID: https://orcid.org/0000-0001-8492-2499)
- Peng Zhang (ORCID: https://orcid.org/0000-0002-6881-9800)
- Zhongyu Li (ORCID: https://orcid.org/0000-0002-9566-5771)
- Luo Liu (ORCID: https://orcid.org/0000-0002-3542-8213)
Institutions
- Nanjing Tech University (CN)
- Xiamen University (CN)
- Czech Academy of Sciences, Institute of Biotechnology (CZ)
- DWI – Leibniz Institute for Interactive Materials (DE)
- Leibniz Institute of Plant Biochemistry (DE)
- Bio-oriented Technology Research Advancement Institution (JP)
- Innovation Research Center (US)
- Nano Carbon (Poland) (PL)
- State Key Laboratory of Materials-Oriented Chemical Engineering
- Beijing University of Chemical Technology (CN)
- RWTH Aachen University (DE)
Publication Details
- Journal
- ACS Sustainable Chemistry & Engineering
- Published
- 2025-03-06
- DOI
- https://doi.org/10.1021/acssuschemeng.4c05423
- Citations
- 2
- Primary Topic
- Computational Drug Discovery Methods
- Type
- article
- Field-Weighted Citation Impact
- 3.01
Funders
- National Natural Science Foundation of China
- Horizon 2020 Framework Programme
- National Key Research and Development Program of China