Molecular determinants for selective C-25-hydroxylation of vitamins D-2 and D-3 by fungal peroxygenases

Hydroxylation of vitamin D by Agrocybe aegerita and Coprinopsis cinerea peroxygenases was investigated in a combined experimental and computational study. 25-Monohydroxylated vitamins D-3 (cholecalciferol) and D-2 (ergocalciferol), compounds of high interest in human health and animal feeding, can be obtained through a reaction with both fungal enzymes. Differences in conversion rates, and especially in site selectivity, were observed. To rationalize the results, diffusion of D-2 and D-3 on the molecular structure of the two enzymes was performed using the PELE software. In good agreement with experimental conversion yields, simulations indicate more favorable energy profiles for the substrates' entrance in C. cinerea than for A. aegerita enzyme. On the other hand, GC-MS analyses show that while a full regioselective conversion of D-2 and D-3 into the active C-25 form is catalyzed by C. cinerea peroxygenase, A. aegerita yielded a mixture of the hydroxylated D-3 products. From the molecular simulations, relative distance distributions between the haem compound I oxygen atom and H-24/H-25 atoms (hydrogens on C-24 and C-25, respectively) were plotted. Results show large populations for O-H-25 distances below 3 angstrom for D-2 and D-3 in C. cinerea in accordance with the high reactivity observed for this enzyme. In A. aegerita, however, cholecalciferol has similar populations (below 3 angstrom) for O-H-25 and O-H-24, which can justify the hydroxylation observed in C-24. In the case of ergocalciferol, due to the bulky methyl group in position C-24, very few structures are found with O-H-24 distances below 3 angstrom and thus, as expected, the reaction was only observed at the C-25 position.