Geometry-encoded molecular dynamics enables deep learning insights into P450 regiospecificity control.

Cytochrome P450 1A2, as many isoenzymes, can generate multiple metabolites from a single substrate. A loose coupling between substrate binding and oxygen activation makes possible substrate reorientations at the active site prior to catalysis. In the present work, caffeine oxidation to alternative bioactive compounds was used to decipher this pluripotency. A model involving two interacting subsites capable of sequentially accommodating one or two caffeine molecules was considered. Molecular dynamics was used to characterize subsite interactions and feed a dedicated geometric encoding of trajectories that was coupled to dimensional reductions and differential machine learning. The two subsites differentially control caffeine orientations and can exchange substrate through a phenylalanine gated mechanism. This exchange can be locked by the presence of a second bound molecule. Complementary roles of subsites in progressively determining the caffeine orientation during its approach to active oxygen were examined. Interestingly, substrate face flipping becomes impaired upon entry into the rather flat active site. This makes the mechanisms that define the orientation of caffeine relative to active oxygen dependent on the substrate face oriented toward heme. Globally, this evidenced that P450 1A2 regioselectivity results from local determinants combined with subsite interactions and caffeine face preselection at a longer distance.