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Stabilization of the Reductase Domain in the Catalytically Self‐Sufficient Cytochrome P450 BM3 by Consensus‐Guided Mutagenesis
Author(s) -
SaabRincón Gloria,
Alwaseem Hanan,
GuzmánLuna Valeria,
Olvera Leticia,
Fasan Rudi
Publication year - 2018
Publication title -
chembiochem
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.05
H-Index - 126
eISSN - 1439-7633
pISSN - 1439-4227
DOI - 10.1002/cbic.201700546
Subject(s) - mutagenesis , protein engineering , reductase , enzyme , chemistry , bacillus megaterium , saturated mutagenesis , directed evolution , site directed mutagenesis , rational design , biochemistry , thermostability , stereochemistry , mutation , biology , gene , mutant , genetics , bacteria
The multidomain, catalytically self‐sufficient cytochrome P450 BM‐3 from Bacillus megaterium (P450 BM3 ) 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 P450 BM3 . 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 P450 BM3 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 P450 BM3 variants developed here could potentially provide more robust scaffolds for the engineering of oxidation biocatalysts.