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Self-sufficient P450-reductase chimeras for biocatalysis
1Skaggs Doctoral Program in the Chemical and Biological Sciences, Scripps Research, La Jolla, CA, United States.
Methods in Enzymology
|November 17, 2023
Summary
Engineered self-sufficient cytochromes P450 were created by fusing P450 enzymes with reductase domains. These novel biocatalysts enable efficient, site-selective functionalization of small molecules for diverse chemical applications.
Area of Science:
- Biocatalysis and enzyme engineering
- Organic chemistry
- Synthetic biology
Background:
- Cytochromes P450 are versatile biocatalysts for chemical transformations.
- Most P450s require redox partners, limiting their application.
- Self-sufficient P450s offer a model for engineered systems.
Purpose of the Study:
- To engineer self-sufficient P450s by fusing P450s with reductase domains.
- To create artificial P450-reductase fusions for enhanced biocatalysis.
- To demonstrate the applicability of this approach for diverse P450s.
Main Methods:
- Designing and assembling artificial P450-reductase chimeras.
- Covalently linking Streptomyces P450s (PtmO5, TleB) with the P450RhF reductase domain.
- Verifying enzyme activity and applying them in preparative-scale reactions.
Main Results:
- Successfully generated two engineered, self-sufficient P450 enzymes.
- Confirmed the biocatalytic activity of the engineered fusion proteins.
- Utilized the enzymes in preparative-scale reactions for chemical synthesis.
Conclusions:
- The fusion approach enables the creation of self-sufficient P450 biocatalysts.
- This method is applicable to a wide range of P450 enzymes.
- Engineered self-sufficient P450s have broad potential in chemical applications.
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