P450 engineering via structure-guided rational design achieves high C21-selectivity and bioconversion in steroid...

Area of Science:

• Biocatalysis and Enzyme Engineering
• Synthetic Chemistry
• Molecular Biology

Background:

• C21-hydroxylation is a critical step in synthesizing corticosteroids like cortisol and aldosterone.
• The enzyme CYP154C5 from Nocardia farcinica is a potential candidate for this biotransformation.
• Engineering P450 enzymes is essential for improving their efficiency and selectivity in steroid synthesis.

Purpose of the Study:

• To engineer a highly efficient and regioselective C21-hydroxylase for progesterone (PRO) conversion.
• To enhance the catalytic activity and substrate scope of the engineered enzyme.
• To elucidate the structural basis for the observed regioselectivity shift using computational methods.

Main Methods:

• Structure-guided rational design and focused rational iterative site-directed mutagenesis (FRISM) were employed to engineer wild-type CYP154C5.
• A redox fusion variant (M6a-RhFRED L3) was constructed to enhance catalytic performance.
• Molecular docking and molecular dynamics (MD) simulations were used to analyze substrate binding and active site dynamics.

Main Results:

• The engineered variant M6a exhibited a complete shift in regioselectivity from C16α to C21 hydroxylation of PRO, with 98% selectivity and >99% conversion.
• The M6a-RhFRED L3 variant showed a 1.43-fold improvement in catalytic activity compared to M6a-RhFRED.
• M6a-RhFRED L3 successfully catalyzed the C21-hydroxylation of three steroid analogs with 98% selectivity and >60% conversion.

Conclusions:

• The engineered M6a variant and its redox fusion derivative M6a-RhFRED L3 are highly efficient biocatalysts for steroid C21-hydroxylation.
• Key residue modifications are crucial for modulating P450 enzyme regioselectivity, enabling a shift from C16α to C21 hydroxylation.
• This study provides valuable insights for the rational engineering of P450 enzymes for industrial applications in steroid synthesis.