Laboratory evolution of P450 BM-3 for mediated electron transfer

Area of Science:

• Biocatalysis
• Enzyme Engineering
• Synthetic Chemistry

Background:

• Cell-free synthesis using P450 monooxygenases is limited by expensive cofactor requirements like NAD(P)H.
• Developing cost-effective and efficient cofactor systems is crucial for advancing preparative synthesis.

Purpose of the Study:

• To design and validate a medium-throughput screening system for improving P450 monooxygenases.
• To establish zinc/cobalt(III)sepulchrate (Zn/Co(III)sep) as an alternative cofactor system for mediated electron transfer.
• To identify enhanced P450 variants with improved catalytic efficiency.

Main Methods:

• Development of a 96-well format screening system utilizing Zn/Co(III)sep for mediated electron transfer.
• Employing P450 BM-3 F87A as a model system for optimization and screening.
• Utilizing saturation mutagenesis at amino acid position R47 to generate a mutant library.

Main Results:

• Optimized screening conditions achieved a coefficient of variation below 10%.
• Two double mutants, P450 BM-3(F87A R47F) and P450 BM-3 (F87A R47Y), exhibited 2-3 fold increased catalytic efficiency compared to P450 BM-3 F87A.
• Kinetic analysis revealed that the double mutants' performance was dependent on Co(III)sep concentration, with improved kcat and decreased Km values.

Conclusions:

• The developed screening system effectively identifies P450 variants with enhanced activity using a cost-effective Zn/Co(III)sep cofactor system.
• Mediated electron transfer with Zn/Co(III)sep offers a viable alternative to traditional NAD(P)H-dependent reduction systems for P450-catalyzed synthesis.
• The identified double mutants demonstrate significant improvements in catalytic efficiency, paving the way for more efficient biocatalytic applications.