Structure-Based Engineering of Steroidogenic CYP260A1 for Stereo- and Regioselective Hydroxylation of Progesterone

Abstract

The production of regio- and stereoselectively hydroxylated steroids is of high pharmaceutical interest and can be achieved by cytochrome P450-based biocatalysts. CYP260A1 from Sorangium cellulosum strain So ce56 catalyzes hydroxylation of C19 or C21 steroids at the very unique 1α-position. However, the conversion of progesterone (PROG) by CYP260A1 is very unselective. In order to improve its selectivity we applied a semirational protein engineering approach, resulting in two different, highly regio- and stereoselective mutants by replacing a single serine residue (S276) of the substrate recognition site 5 with an asparagine or isoleucine. The S276N mutant converted PROG predominantly into 1α-hydroxy-PROG, while the S276I mutant led to 17α-hydroxy-PROG. We solved the high-resolution crystal structures of the PROG-bound S276N and S276I mutants, which revealed two different binding modes of PROG in the active site. The orientations were consistent with the exclusive 1α- (pro-1α binding mode) and 17α-hydroxylation (pro-17α-binding mode) of S276N and S276I, respectively. We observed that water-mediated hydrogen bonds contribute to the stabilization of the polar C3 and C17 substituents of PROG. Both binding modes of PROG may be stabilized in the wild-type enzyme. The change in regioselectivity is mainly driven by destabilizing the alternative binding mode due to steric hindrance and hydrogen bond disruption, caused by the mutations of Ser276. Thus, for the first time, the change in the selectivity of cytochrome P450-mediated steroid hydroxylation created by rational mutagenesis can be explained by the obtained 3D structures of the substrate-bound mutants, providing the basis for further experiments to engineer the biocatalyst toward novel steroid hydroxylation positions.

Bacterial cytochrome P450-catalyzed regio- and stereoselective steroid hydroxylation enabled by directed evolution and rational design

“…Structure-guided rational design was conducted to mutate S326 to an asparagine, resulting in mutant S326R that displayed much better activity and selectivity for the formation of Best CYP106A2 mutants on progesterone. Relative values [%] of progesterone substrate and monohydroxylated products (15β-, 11α-, 9α-and 6β) produced by CYP106A2 WT mutants A395I and G395W/G397K, and best combinatorially derived mutants in 6 h whole-cell biotransformations with 200 μM of progesterone (Nguyen et al 2012) 1α-hydroxy-11-deoxycorticosterone compared to the WT parent (Khatri et al 2018).…”

“…In order to improve its regio-and stereoselectivity for progesterone transformation, Khatri et al cloned and expressed truncated protein of CYP260A1 (∆CYP260A1) (Khatri et al 2018), and the first 50 N-terminal residues were removed based on the alternative predicted gene transcript from the NCBI database (Khatri et al 2016a, b). Laborious efforts combining rational design and directed evolution were employed to create mutants of ∆CYP260A1 with improved regioselectivity towards progesterone hydroxylation (Khatri et al 2018). After recovery of complete activity of ∆CYP260A1, the substrate was docked into ∆CYP260A1 active site to explore potential hotspot residues.…”

“…Relative values in % of progesterone and its monohydroxylated products 15β-, 11α-, 9α-and 6β-hydroxyprogesterone after 24 h in vivo conversion of 200 μM progesterone catalyzed by WT CYP106A2 and mutant proteins thereof. Values show percentages with the sum of the substrate progesterone and all monohydroxylated products set as 100% (n.d.*: not detectable) (Nikolaus et al 2017 (Khatri et al 2018). With the addition of position 17α to the above targeted positions, it is clear that directed evolution can generate highly regio-and stereoselective enzymes for challenging biotransformation.…”

“…Engineering CYP260A1 for progesterone functionalization at positions 1α and 17α. Strategy for constructing mutants of CYP260A1 and experimental workflow(Khatri et al 2018) References Acevedo-Rocha CG, Gamble C, Lonsdale R, Li A, Nett N, Hoebenreich S, Deege A (2018) P450-catalyzed regio-and diastereoselective steroid hydroxylation: efficient directed evolution enabled by mutability landscaping. ACS Catal 8(4):3395-3410 Agematu H, Matsumoto N, Fujii Y, Kabumoto H, Doi S, Machia K, Arisawa A (2006) Hydroxylation of testosterone by bacterial cytochromes P450 using the Escherichia coli expression system.…”

P450 Monooxygenases Enable Rapid Late‐Stage Diversification of Natural Products via C−H Bond Activation

“…Such a map of the sequence‐function relationship was generated for the F87A template enzyme by analysing previous data from Commandeur et al ., Payne et al ., and their own lab, and by screening mutants available from a previous study . For example the group of Commandeur had identified residue 72 to be important for substrate orientation, and showed that it was completely inverting the stereoselectivity of C16 hydroxylation .…”