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P450BM3 fused to phosphite dehydrogenase allows phosphite-driven selective oxidations
Nina Beyer1, Justyna K Kulig2, Anette Bartsch1
1Department of Biochemistry, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Nijenborgh 4, 9747 AG, Groningen, The Netherlands.
Applied Microbiology and Biotechnology
|December 1, 2016
Summary
Researchers created a self-sufficient enzyme by fusing P450BM3 with phosphite dehydrogenase (PTDH). This PTDH-P450BM3 fusion enzyme efficiently regenerates NADPH using phosphite, enhancing biocatalysis and drug metabolism studies.
Area of Science:
- Biocatalysis
- Enzyme Engineering
Background:
- NADPH-dependent P450BM3 is a powerful enzyme but requires cofactor regeneration.
- NADPH regeneration often relies on external systems, limiting broader application.
Purpose of the Study:
- To develop a self-sufficient P450BM3 system for enhanced biocatalysis.
- To create a fusion enzyme that utilizes phosphite for NADPH regeneration.
Main Methods:
- Genetic fusion of P450BM3 monooxygenase with phosphite dehydrogenase (PTDH).
- Purification and characterization of the PTDH-P450BM3 fusion enzyme.
- Assessing catalytic activity and cofactor regeneration using lauric acid, omeprazole, and rosiglitazone as substrates.
Main Results:
- The PTDH-P450BM3 fusion enzyme demonstrated efficient NADPH regeneration using phosphite.
- The fusion enzyme exhibited higher activity and comparable substrate selectivity to native P450BM3.
- PTDH-P450BM3 showed improved efficiency in drug compound conversions compared to NADPH-dependent P450BM3.
Conclusions:
- The PTDH-P450BM3 fusion enzyme is a self-sufficient and efficient biocatalyst.
- This system offers advantages for biocatalytic applications and drug metabolism studies.
- The fusion approach enhances enzyme performance and cofactor utilization.
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