Title
Stabilization of the Reductase Domain in the Catalytically Self-Sufficient Cytochrome P450BM3 via Consensus-Guided Mutagenesis
Author
Gloria Saab-Rincon, Hanan Alwaseem, Valeria Guzmán-Luna, Leticia Olvera, Rudi Fasan
Year
2018
Journal
ChemBioChem
Abstract
The multi-domain, catalytically self-sufficient cytochrome P450 BM-3 from Bacillus megaterium (P450BM3) constitutes a versatile enzyme for the oxyfunctionalization of organic molecules and natural products. However, the limited stability of the diflavin reductase domain limits the utility of this enzyme for synthetic applications. In this work, a consensus-guided mutagenesis approach was applied to enhance the thermal stability of the reductase domain of P450BM3. Upon phylogenetic analysis of a set of distantly related P450s (% identity > 38%), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effect relative to the wild-type reductase domain. Recombination of the six most stabilizing mutations resulted in the generation of two thermostable variants featuring up to 10-fold longer half-lives at 50°C and increased catalytic performance at elevated temperature. Further characterization of the engineered P450BM3 variants indicated that the introduced mutations increase the thermal stability of the FAD-binding domain and that the optimal temperature (Topt) of the enzyme has shifted from 25°C to 40°C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multi-domain P450. The stabilized P450BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.
Instrument
J-715
Keywords
Circular dichroism, Secondary structure, Tertiary structure, Thermal stability, Thermodynamics, Biochemistry