Stabilization of the Reductase Domain in the Catalytically Self‐Sufficient Cytochrome P450BM3 by Consensus‐Guided Mutagenesis. (12th February 2018)

• Record Type: Journal Article
• Authors: Saab‐Rincón, Gloria; Alwaseem, Hanan; Guzmán‐Luna, Valeria; Olvera, Leticia; Fasan, Rudi
• Is Part Of: Chembiochem. Volume 19:Number 6(2018)
• Page Start: 622
• Page End: 632
• Publication Date: 2018-02-12
• Subjects: consensus mutagenesis -- CYP102A1 -- cytochrome P450 -- enzyme thermostabilization -- P450 BM-3; Biochemistry -- Periodicals; Molecular biology -- Periodicals; Pharmaceutical chemistry -- Periodicals; 572
• DOI: 10.1002/cbic.201700546
• Languages: English
• ISSNs: 1439-4227

Abstract

Abstract: The multidomain, 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 (>38 % identity), a total of 14 amino acid substitutions were identified and evaluated in terms of their stabilizing effects relative to the wild‐type reductase domain. Recombination of the six most stabilizing mutations generated two thermostable variants featuring up to tenfold longer half‐lives at 50 °C and increased catalytic performance at elevated temperatures. Further characterization of the engineered P450BM3 variants indicated that the introduced mutations increased the thermal stability of the FAD‐binding domain and that the optimal temperature ( T opt ) of the enzyme had shifted from 25 to 40 °C. This work demonstrates the effectiveness of consensus mutagenesis for enhancing the stability of the reductase component of a multidomain P450. The stabilized P450BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts. Abstract : Handling the heat : Consensus‐guided mutagenesis was applied to stabilize the reductase domain of the biotechnologically useful P450BM3 enzyme. The evolved variants exhibited improved robustness to thermal denaturation and increased catalytic performance at elevated temperatures.