title: Steroid hydroxylation by basidiomycete peroxygenases: a combined experimental and computational study.
abstract: The 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 H2O2 as the only cosubstrate. Two of them are wild-type enzymes from Agrocybe aegerita and Marasmius rotula, and the third one is a recombinant enzyme from Coprinopsis 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.
authors: Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Seville, Spain.
authors: Esteban D
authors: ED
authors: Babot
authors: Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Seville, Spain.
authors: José C
authors: JC
authors: Del Río
authors: Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Barcelona, Spain Anaxomics Biotech, Barcelona, Spain.
authors: Marina
authors: M
authors: Cañellas
authors: Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Barcelona, Spain Anaxomics Biotech, Barcelona, Spain.
authors: Ferran
authors: F
authors: Sancho
authors: Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Barcelona, Spain.
authors: Fátima
authors: F
authors: Lucas
authors: Joint BSC-CRG-IRB Research Program in Computational Biology, Barcelona Supercomputing Center, Barcelona, Spain ICREA, Barcelona, Spain.
authors: Víctor
authors: V
authors: Guallar
authors: Novozymes A/S, Bagsvaerd, Denmark.
authors: Lisbeth
authors: L
authors: Kalum
authors: Novozymes A/S, Bagsvaerd, Denmark.
authors: Henrik
authors: H
authors: Lund
authors: JenaBios GmbH, Jena, Germany.
authors: Glenn
authors: G
authors: Gröbe
authors: JenaBios GmbH, Jena, Germany.
authors: Katrin
authors: K
authors: Scheibner
authors: TU Dresden, Department of Bio- and Environmental Sciences, Zittau, Germany.
authors: René
authors: R
authors: Ullrich
authors: TU Dresden, Department of Bio- and Environmental Sciences, Zittau, Germany.
authors: Martin
authors: M
authors: Hofrichter
authors: Centro de Investigaciones Biológicas, CSIC, Madrid, Spain.
authors: Angel T
authors: AT
authors: Martínez
authors: Instituto de Recursos Naturales y Agrobiología de Sevilla, CSIC, Seville, Spain anagu@irnase.csic.es.
authors: Ana
authors: A
authors: Gutiérrez
mesh_terms: D000363:Agaricales / Q000201:enzymology / Q000378:metabolism*; D002849:Chromatography, Gas; D003198:Computer Simulation; D006861:Hydrogen Peroxide / Q000378:metabolism; D006900:Hydroxylation; D007659:Ketones / Q000378:metabolism; D055433:Marasmius / Q000201:enzymology / Q000378:metabolism*; D013058:Mass Spectrometry; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D013237:Stereoisomerism; D013256:Steroids / Q000737:chemistry* / Q000378:metabolism*
chemical_list: D007659:Ketones; D013256:Steroids; D006861:Hydrogen Peroxide; D006899:Mixed Function Oxygenases; C022333:peroxygenase