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Novel approach to improve progesterone hydroxylation selectivity by CYP 106A2 via rational design of adrenodoxin binding
Author(s) -
Sagadin Tanja,
Riehm Jan,
Putkaradze Natalia,
Hutter Michael C.,
Bernhardt Rita
Publication year - 2019
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/febs.14722
Subject(s) - adrenodoxin , hydroxylation , chemistry , selectivity , bacillus megaterium , mutant , docking (animal) , rational design , biochemistry , stereochemistry , enzyme , cytochrome p450 , gene , biology , genetics , catalysis , bacteria , medicine , nursing
Bacterial P450s have considerable potential for biotechnological applications. The P450 CYP 106A2 from Bacillus megaterium ATCC 13368 converts progesterone to several hydroxylated products that are important precursors for pharmaceutical substances. As high yields of monohydroxylated products are required for biotechnological processes, improving this conversion is of considerable interest. It has previously been shown that the binding mode of the redox partner can affect the selectivity of the progesterone hydroxylation, being more stringent in case of the Etp1 compared with Adx(4–108). Therefore, in this study we aimed to improve hydroxylation selectivity by optimizing the binding of Adx(4–108) with CYP 106A2 allowing for a shorter distance between both redox centers. To change the putative binding interface of Adx(4–108) with CYP 106A2, molecular docking was used to choose mutation sites for alteration. Mutants at positions Y82 and P108 of Adx were produced and investigated, and confirmed our hypothesis. Protein–protein docking, as well as conversion studies, using the mutants demonstrated that the iron–sulfur(FeS) cluster/heme distance diminished significantly, which subsequently led to an approximately 2.5‐fold increase in 15β‐hydroxyprogesterone, the main product of progesterone conversion by CYP 106A2.