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The so-called dye-decolorizing peroxidases (DyPs) constitute a new family of proteins exhibiting remarkable stability. With the aim of providing them new catalytic activities of biotechnological interest, the heme pocket of one of the few DyPs fully characterized to date (from the fungus Auricularia auricula-judae) was redesigned based on the crystal structure available, and its potential for asymmetric sulfoxidation was evaluated. Chiral sulfoxides are important targets in organic synthesis and enzyme catalysis, due to a variety of applications. Interestingly, one of the DyP variants, F359G, is highly stereoselective in sulfoxidizing methyl-phenyl sulfide and methyl-p-tolyl sulfide (95–99% conversion, with up to 99% excess of the Senantiomer in short reaction times), while the parent DyP has no sulfoxidation activity, and the L357G variant produces both Rand Senantiomers. The two variants were crystallized, and their crystal structures were used in molecular simulations to provide a rational explanation for the new catalytic activities. Protein energy landscape exploration (PELE) showed more favorable protein–substrate catalytic complexes for the above variants, with a considerable number of structures near the oxygen atom of the activated heme, which is incorporated into the substrates as shown in 18O-labeling experiments, and improved affinity with respect to the parent enzyme, explaining their sulfoxidation activity. Additional quantum mechanics/molecular mechanics (QM/MM) calculations were performed to elucidate the high stereoselectivity observed for the F359G variant, which correlated with higher reactivity on the substrate molecules adopting pro-Sposes at the active site. Similar computational analyses can help introduce/improve (stereoselective) sulfoxidation activity in related hemeproteins.

Hydroxylation of vitamin D by Agrocybe aegeritaand Coprinopsis cinereaperoxygenases 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 D2and D3on 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. cinereathan for A. aegeritaenzyme. On the other hand, GC-MS analyses show that while a full regioselective conversion of D2and D3into the active C25form is catalyzed by C. cinereaperoxygenase, A. aegeritayielded a mixture of the hydroxylated D3products. From the molecular simulations, relative distance distributions between the haem compound I oxygen atom and H24/H25atoms (hydrogens on C24and C25, respectively) were plotted. Results show large populations for O–H25distances below 3 Å for D2and D3in C. cinereain accordance with the high reactivity observed for this enzyme. In A. aegerita, however, cholecalciferol has similar populations (below 3 Å) for O–H25and 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–H24distances below 3 Å and thus, as expected, the reaction was only observed at the C25position.