Speaker
Description
Title:
Traceless cofactor regeneration from formate for CYP450 enzyme cascades
Authors:
Ping Xiao (1), Jibira Yakubu (2), Amit V Pandey (2), Christoph von Ballmoos (1)
Affiliations
(1) University of Bern, Department of Chemistry, Biochemistry and Pharmacy, Freiestrasse 3, 3012 Bern, Switzerland
(2) University of Bern, Department for BioMedical Research, Murtenstrasse 28, CH-3008 Bern, Switzerland
Abstract:
Background: Cytochrome P450 enzymes are essential for the metabolism of clinically relevant drugs and for physiological processes such as hormone biosynthesis. Their remarkable catalytic versatility has made them highly valuable in applications ranging from personalized medicine and drug development to synthetic biology. Numerous in vitro platforms, such as liver microsomes, whole-cell systems, and purified enzyme preparations, have been developed to investigate CYP450 catalysis. A common feature of these systems is their dependence on a continuous supply of NADPH as the primary electron donor. However, this is a major limitation because external NADPH is costly and unstable, and NADPH regeneration systems can produce byproducts that may affect CYP450 activity and complicate product purification.
Method: Using formate dehydrogenase (FDH) and soluble transhydrogenase (SthA), we present a two-enzyme system that continuously supplies NADPH from low-cost formate to drive CYP450 catalysis, while producing CO₂ as the only byproduct that readily leaves the solution as a gas.
Results: We successfully expressed and purified FDH and SthA, and then systematically evaluated their individual activities in different buffer systems, and the effects of temperature, pH, and metal ions on their catalytic activity. We next examined their coupling efficiency and substrate conversion. Finally, the NADPH regeneration system was applied to support CYP450 protein cascades.
Conclusion: We successfully developed an FDH- and SthA-based two-enzyme NADPH regeneration system that enables continuous CYP450 catalysis using low-cost formate. Its efficiency and compatibility with different CYP450 platforms highlight its potential for future applications in biocatalysis, synthetic biology, and pharmaceutical development.
Acknowledgments:
Supported by Swiss National Science Foundation (SNSF).