Changing the regioselectivity of a P450 from C15 to C11 hydroxylation of progesterone

Abstract

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.