Steroid hydroxylation by basidiomycete peroxygenases: a combined experimental and computational study

Poster nº 40 presentado en el 4th New Trends in Computational Chemistry for Industrial Applications 1-2 Octubre, Barcelona (2015)Steroids comprise a group of cyclic organic compounds with a four-ring structure. They represent an important class of natural compounds that are widely spread in the nature underlying structural functions and many biological processes. Moreover, steroids have a huge variety of pharmacological properties, being the second largest category among medical products. The structure of these compounds has a direct relation with the physiological activity2. Thus, steroids can be naturally modified using fungal enzymes in order to search for new and more active compounds3. With this purpose, peroxygenases from three basidiomycete species were tested for the hydroxylation of a variety of steroidal compounds, using H2O2 as the only cosubstrate. 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 recently4. 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 activesite 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.(1) Babot ED, del Río JC, Cañellas M, Sancho F, Lucas F, Guallar V, Kalum L, Lund H, Gröbe G, Scheibner K, Ullrich R, Hofrichter M, Martínez AT, Gutiérrez A, Appl Environ Microbiol 2015, 81, 4130 –4142. (2) Donova MV, Egorova OV, Appl Microbiol Biotechnol, 2012, 94, 1423–1447. (3) Beneventi E, Ottolina G, Carrea G, Panzeri W, Fronza G, Lau PCK. 2009. Enzymatic Baeyer-Villiger oxidation of steroids with cyclopentade- canone monooxygenase. J Mol Catal B Enzym 58:164 –168. (4) Blanc, M et al., Immunity 2013, 38,106 –118Funded by the INDOX (KBBE-2013-7-613549) European Project.Peer Reviewe