Abstract
Steroids are the most widely marketed products by the pharmaceutical industry after antibiotics. Steroid hydroxylation is one of the most important functionalizations because their derivatives enable a higher biological activity compared to their less polar non-hydroxylated analogs. Bacterial cytochrome P450s constitute promising biocatalysts for steroid hydroxylation due to their high expression level in common workhorses like Escherichia coli. However, they often suffer from wrong or insufficient regio- and/or stereoselectivity, low activity, narrow substrate range as well as insufficient thermostability, which hampers their industrial application. Fortunately, these problems can be generally solved by protein engineering based on directed evolution and rational design. In this work, an overview of recent developments on the engineering of bacterial cytochrome P450s for steroid hydroxylation is presented.
Highlights
Cytochrome P450 (CYPs) belong to a superfamily of heme-containing enzymes which typically act as monooxygenase, catalyzing the reductive scission of molecular oxygen, thereby introducing one oxygen atom into the substrate, whereas the second oxygen atom is reduced to water (Urlacher and Girhard 2012)
Directed evolution based on combinatorial active-site saturation test (CAST), iterative saturation mutagenesis (ISM), mutability landscapes (MLs), molecular dynamics simulation (MDs), etc. (Kille et al 2011)
In this review, we summarized the achievements attained in the engineering of bacterial cytochrome P450 monooxygenases (CYPs) towards steroid hydroxylation during the past decades
Summary
Cytochrome P450 (CYPs) belong to a superfamily of heme-containing enzymes which typically act as monooxygenase, catalyzing the reductive scission of molecular oxygen, thereby introducing one oxygen atom into the substrate, whereas the second oxygen atom is reduced to water (Urlacher and Girhard 2012). Mutagenesis was started on sites A (R47/T49/ Y51), B (V78/A82) and C (M185/L188) using NDC, Table 1 Summary of CYPs variants-catalyzed regio- and stereoselective steroid hydroxylation by directed evolution This directed evolution strategy demonstrated how iterative saturation mutagenesis (ISM) enabled the hydroxylation of two steroids with high regio- and stereoselectivity.
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