Vitamin D₃ Activation by Cytochrome P450 Enzymes: Differences between Bacterial and Human Calcitriol Biosynthesis

Vitamin D₃ (VD₃) is an important natural product with functions in the human body related to bone growth and homeostasis. In the body, vitamin D₃ is converted into the hormone calcitriol by successive activation of the C₂₅-H and C₁-H groups by two cytochrome P450 isozymes that each catalyze a regioselective C-H hydroxylation step. By contrast, in bacteria, both reactions happen in the same P450 isozyme, but the product after the first hydroxylation step does not escape into solution. To understand the differences between human and bacterial VD₃ activation, we performed molecular dynamics and quantum mechanics studies of these enzymes. Molecular dynamics simulations on the four P450 structures for human and bacterial isozymes with VD₃ and 25-hydroxy-VD₃ bound show that the human P450 enzyme for the first hydroxylation step has a small and narrow cavity around the heme, which only fits an aliphatic chain, and hence can only perform C₂₅-hydroxylation. On the other hand, the bacterial P450 has a more open and spherical substrate binding pocket where the substrate fits in both orientations. Consequently, 25-hydroxy-VD₃ will bind for a long period of time in the bacterial P450 isozyme that can trigger a second reaction cycle with molecular oxygen. Subsequently, QM calculations on cluster models show that all reaction steps happen through a rate-determining hydrogen atom abstraction. However, the human P450 isozyme reacts with faster kinetics than the bacterial isozyme through better and tighter positioning of the substrate, which highlights the role of the second coordination sphere for enzyme-catalyzed reactions.