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Changing the Regioselectivity of a P450 from C15 to C11 Hydroxylation of Progesterone
Author(s) -
Nguyen Kim Thoa,
Virus Cornelia,
Günnewich Nils,
Hannemann Frank,
Bernhardt Rita
Publication year - 2012
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.201100811
Subject(s) - saturated mutagenesis , hydroxylation , regioselectivity , mutant , chemistry , mutagenesis , directed evolution , site directed mutagenesis , active site , steroid , stereochemistry , enzyme , biochemistry , catalysis , gene , hormone
CYP106A2 is known as a 15β‐hydroxylase, but also shows minor 11α‐hydroxylase activity for progesterone. 11α‐Hydroxyprogesterone is an important pharmaceutical compound with anti‐androgenic and blood‐pressure‐regulating activity. This work therefore focused on directing the regioselectivity of the enzyme towards hydroxylation at position 11 in the C ring of the steroid through a combination of saturation mutagenesis and rational site‐directed mutagenesis. With the aid of data from a homology model of CYP106A2 containing docked progesterone, together with site‐directed mutagenesis of active‐site residues (Lisurek et al. ChemBioChem 2008 , 9 , 1439–1449), a saturation mutagenesis library at positions A395 and G397 was created. Screening of the library identified the mutants A395I and A395W/G397K as having 11α‐hydroxylase activities 8.9 and 11.5 times higher than that of the wild type (WT). In the next step, additional mutations were integrated by a rational site‐directed mutagenesis approach to increase the catalytic efficiency. Of the 40 candidates analyzed, the mutants A106T/A395I, A106T/A395I/R409L, and T89N/A395I turned out to display increased 11α‐hydroxylase selectivities and activities relative to the WT (14.3‐, 12.6‐, and 11.8‐fold increases in selectivity and 39.3‐, 108‐, and 24.4‐ in k cat / K m ). In the last step of the study, the best mutants were applied in a whole‐cell biotransformation. In these experiments the production (percentage) of 15β‐hydroxyprogesterone decreased from 50.4 % (wild type) to 4.8 % (mutant T89N/A395I), whereas that of 11α‐hydroxyprogesterone increased from 27.7 to 80.9 %, thus demonstrating an impressive regioselectivity.