A Novel A105Y Mutant\nof CYP17A1 Exhibits Almost Perfect\nRegioselectivity in the Production of 17α-Hydroxyprogesterone

17α-Hydroxyprogesterone\n(17α-OHP) is a steroid hormone\nwith significant biological activity that can be obtained by catalyzing\nprogesterone (PROG), the main product of sitosterol, through CYP17A1.\nHowever, increasing the catalytic specificity of HCYP17A1 for C17\nhydroxylation of progesterone (PROG) poses a formidable challenge\ndue to the close proximity of the C16 and C17 positions. In this study,\na rational design was utilized to alter the spatial configuration\nof the substrate channel, leading to the complete abolition of its\n16-hydroxylation activity. Subsequent molecular dynamics simulations\nrevealed that the A105Y mutation heightened the rigidity of the G95–I112\nregion of CYP17A1, consequently regulating the direction of the entry\nof PROG into the catalytic pocket. Moreover, the establishment of\nhydrogen bonding between Y105 and R239, coupled with Pi-stacking of\nA105Y with F114, effectively immobilizes the substrate PROG in a fixed\nposition, explaining the practically perfect regioselectivity observed\nin A105Y. Finally, a multifaceted enzymatic cascade system, incorporating\nA105Y, cytochrome P450 reductase (CPR), and glucose-6-phosphate dehydrogenase\n(ZWF) for NADPH cofactor regeneration, was constructed in Pichia pastoris GS115. The resulting biocatalyst\nproduced 106 ± 3.2 mg L<sup>–1</sup> 17α-OHP, a\n4.6-fold increase compared with A105Y alone. Thus, this study provides\nvaluable insights for improving the regioselectivity and activity\nof P450 enzymes.