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Molecular Evolution of a Steroid Hydroxylating Cytochrome P450 Using a Versatile Steroid Detection System for Screening
Author(s) -
Virus Cornelia,
Bernhardt Rita
Publication year - 2008
Publication title -
lipids
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.601
H-Index - 120
eISSN - 1558-9307
pISSN - 0024-4201
DOI - 10.1007/s11745-008-3236-8
Subject(s) - adrenodoxin , hydroxylation , cytochrome p450 , directed evolution , steroid , biochemistry , mutagenesis , chemistry , cytochrome , bacillus megaterium , enzyme , cyp17a1 , biology , mutation , gene , genetics , bacteria , mutant , hormone
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.