Molecular determinants for selective C25-hydroxylation of vitamins D2 and D3 by fungal peroxygenases

Abstract

Hydroxylation of vitamin D by Agrocybe aegerita and Coprinopsis cinerea peroxygenases was investigated in a combined experimental and computational study. 25-Monohydroxylated vitamins D₃ (cholecalciferol) and D₂ (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 D₂ and D₃ 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 D₂ and D₃ into the active C₂₅ form is catalyzed by C. cinerea peroxygenase, A. aegerita yielded a mixture of the hydroxylated D₃ products. From the molecular simulations, relative distance distributions between the haem compound I oxygen atom and H₂₄/H₂₅ atoms (hydrogens on C₂₄ and C₂₅, respectively) were plotted. Results show large populations for O–H₂₅ distances below 3 Å for D₂ and D₃ in C. cinerea in accordance with the high reactivity observed for this enzyme. In A. aegerita, however, cholecalciferol has similar populations (below 3 Å) for O–H₂₅ and O–H₂₄, which can justify the hydroxylation observed in C₂₄. In the case of ergocalciferol, due to the bulky methyl group in position C₂₄, very few structures are found with O–H₂₄ distances below 3 Å and thus, as expected, the reaction was only observed at the C₂₅ position.