@misc{BabotRioCanellasetal., author = {Babot, Esteban D. and R{\´i}o, Jos{\´e} C. del and Ca{\~n}ellas, Marina and Sancho, Ferran and Lucas, F{\´a}tima and Guallar, V{\´i}ctor and Kalum, Lisbeth and Lund, Henrik and Gr{\"o}be, Glenn and Scheibner, Katrin and Ullrich, Ren{\´e} and Hofrichter, Martin and Mart{\´i}nez, Angel T. and Guti{\´e}rrez, Ana}, title = {Steroid hydroxylation by basidiomycete peroxygenases: A combined experimental and computational study}, series = {Applied and Environmental Microbiology}, volume = {81}, journal = {Applied and Environmental Microbiology}, number = {12}, issn = {0099-2240}, doi = {10.1128/AEM.00660-15}, pages = {4130 -- 4142}, 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 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, and steroid hydrocarbons and ketones were followed 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 recently reported). However, hydroxylation in the ring moiety and terminal hydroxylation at the side-chain was also observed in some steroids, the former favored by the absence of oxygenated groups at C3 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 varying reaction yields observed.}, language = {en} } @misc{MartinezRuizDuenasCamareroetal., author = {Martinez, Angel T. and Ruiz-Duenas, Francisco J. and Camarero, Susana and Serrano, Ana and Linde, Dolores and Lund, Henrik and Vind, Jesper and Tovborg, Morton and Herold-Majumdar, Owik M. and Hofrichter, Martin and Liers, Christiane and Ullrich, Ren{\´e} and Scheibner, Katrin and Sannia, Giovanni and Piscitelli, Alessandra and Sener, Mehmet E. and Kilic, Sibel and Berkel, Willem J. H. van and Guallar, V{\´i}ctor and Lucas, Maria F{\´a}tima and Zuhse, Ralf and Ludwig, Roland and Hollmann, Frank and Fern{\´a}ndez-Fueyo, Elena and Record, Eric and Faulds, Craig B. and Tortajada, Marta and Winckelmann, Ib and Rasmussen, Jo-Anne and Gelo-Pujic, Mirjana and Guti{\´e}rrez, Ana and Rio, Jos{\´e} C. del and Rencoret, Jorge and Alcalde, Miguel}, title = {Oxidoreductases on their way to industrial biotransformations}, series = {Biotechnology Advances}, volume = {35}, journal = {Biotechnology Advances}, number = {6}, issn = {1873-1899}, doi = {10.1016/j.biotechadv.2017.06.003}, pages = {815 -- 831}, abstract = {Fungi produce heme-containing peroxidases and peroxygenases, flavin-containing oxidases and dehydrogenases, and different copper-containing oxidoreductases involved in the biodegradation of lignin and other recalcitrant compounds. Heme peroxidases comprise the classical ligninolytic peroxidases and the new dye-decolorizing peroxidases, while heme peroxygenases belong to a still largely unexplored superfamily of heme-thiolate proteins. Nevertheless, basidiomycete unspecific peroxygenases have the highest biotechnological interest due to their ability to catalyze a variety of regio- and stereo-selective monooxygenation reactions with H2O2 as the source of oxygen and final electron acceptor. Flavo-oxidases are involved in both lignin and cellulose decay generating H2O2 that activates peroxidases and generates hydroxyl radical. The group of copper oxidoreductases also includes other H2O2 generating enzymes - copper-radical oxidases - together with classical laccases that are the oxidoreductases with the largest number of reported applications to date. However, the recently described lytic polysaccharide monooxygenases have attracted the highest attention among copper oxidoreductases, since they are capable of oxidatively breaking down crystalline cellulose, the disintegration of which is still a major bottleneck in lignocellulose biorefineries, along with lignin degradation. Interestingly, some flavin-containing dehydrogenases also play a key role in cellulose breakdown by directly/indirectly "fueling" electrons for polysaccharide monooxygenase activation. Many of the above oxidoreductases have been engineered, combining rational and computational design with directed evolution, to attain the selectivity, catalytic efficiency and stability properties required for their industrial utilization. Indeed, using ad hoc software and current computational capabilities, it is now possible to predict substrate access to the active site in biophysical simulations, and electron transfer efficiency in biochemical simulations, reducing in orders of magnitude the time of experimental work in oxidoreductase screening and engineering. What has been set out above is illustrated by a series of remarkable oxyfunctionalization and oxidation reactions developed in the frame of an intersectorial and multidisciplinary European RTD project. The optimized reactions include enzymatic synthesis of 1-naphthol, 25-hydroxyvitamin D3, drug metabolites, furandicarboxylic acid, indigo and other dyes, and conductive polyaniline, terminal oxygenation of alkanes, biomass delignification and lignin oxidation, among others. These successful case stories demonstrate the unexploited potential of oxidoreductases in medium and large-scale biotransformations.}, language = {en} }