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• W1979306972 endingPage "1141" @default.
• W1979306972 startingPage "1133" @default.
• W1979306972 abstract "Molecular evolution is a powerful tool for improving or changing activities of enzymes for their use in biotechnological processes. Cytochromes P450 are highly interesting enzymes for biotechnological purposes because they are able to hydroxylate a broad variety of substrates with high regio- and stereoselectivity. One promising steroid hydroxylating cytochrome P450 for biotechnological applications is CYP106A2 from Bacillus megaterium ATCC 13368. It is one of a few known bacterial cytochromes P450 able to transform steroids such as progesterone and 11-deoxycortisol. CYP106A2 can be easily expressed in Escherichia coli with a high yield and can be reconstituted using the adrenal redox proteins, adrenodoxin and adrenodoxin reductase. We developed a simple screening assay for this system and performed random mutagenesis of CYP106A2, yielding variants with improved 11-deoxycortisol and progesterone hydroxylation activity. After two generations of directed evolution, we were able to improve the k (cat)/K (m) of the 11-deoxycortisol hydroxylation by a factor of more than four. At the same time progesterone conversion was improved about 1.4-fold. Mapping the mutations identified in catalytically improved CYP106A2 variants into the structure of a CYP106A2 model suggests that these mutations influence the mobility of the F/G loop, and the interaction with the redox partner adrenodoxin. The results show the evolution of a soluble steroid hydroxylase as a potential new catalyst for the production of steroidogenic compounds." @default.
• W1979306972 created "2016-06-24" @default.
• W1979306972 creator A5011303922 @default.
• W1979306972 creator A5054561808 @default.
• W1979306972 date "2008-10-01" @default.
• W1979306972 modified "2023-09-23" @default.
• W1979306972 title "Molecular Evolution of a Steroid Hydroxylating Cytochrome P450 Using a Versatile Steroid Detection System for Screening" @default.
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• W1979306972 doi "https://doi.org/10.1007/s11745-008-3236-8" @default.
• W1979306972 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/18830657" @default.
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