Steroid Hydroxylation by Basidiomycete Peroxygenases: a Combined Experimental and Computational Study

ABSTRACTThe goal of this study is the selective oxyfunctionalization of steroids under mild and environmentally friendly conditions using fungal enzymes. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2as the only cosubstrate. Two of them are wild-type enzymes fromAgrocybe aegeritaandMarasmius rotula, and the third one is a recombinant enzyme fromCoprinopsis cinerea. The enzymatic reactions on free and esterified sterols, steroid hydrocarbons, and ketones were monitored by gas chromatography, and the products were identified by mass spectrometry. Hydroxylation at the side chain over the steroidal rings was preferred, with the 25-hydroxyderivatives predominating. Interestingly, antiviral and other biological activities of 25-hydroxycholesterol have been reported recently (M. Blanc et al., Immunity 38:106–118, 2013,http://dx.doi.org/10.1016/j.immuni.2012.11.004). However, hydroxylation in the ring moiety and terminal hydroxylation at the side chain also was observed in some steroids, the former favored by the absence of oxygenated groups at C-3 and by the presence of conjugated double bonds in the rings. To understand the yield and selectivity differences between the different steroids, a computational study was performed using Protein Energy Landscape Exploration (PELE) software for dynamic ligand diffusion. These simulations showed that the active-site geometry and hydrophobicity favors the entrance of the steroid side chain, while the entrance of the ring is energetically penalized. Also, a direct correlation between the conversion rate and the side chain entrance ratio could be established that explains the various reaction yields observed.

Clearance In Vivo of Instilled [H]Cholesterol from the Rat Lung

Phospholipids and lung surfactant proteins are known to be recycled within the lung alveolus mainly by uptake into type II epithelial cells that secrete lipid-enriched lung surfactant. Dipalmitoyl phosphatidylcholine (DPPC) is the major component of lung surfactant lipids and cholesterol is the second most abundant. However, cholesterol turnover in vivo has not been measured and it is not known how long steroidal compounds persist in the lung in intact animals. Here we report on experiments in which radiolabeled cholesterol was instilled into the lungs of rats, then at various postinstillation periods, radioactive sterols in lavage fluid, and in postlavage whole lungs were measured in individual animals. Radioactive sterols in the lungs remained high for a week and were still detectable 46 days later. The clearance rate during the initial postinstillation week was approximately 10% per day. Both radioactive free and esterified sterols were recovered from bronchoalveolar lavage fluid and postlavage lungs.