How experiments and molecular simulations can help understand selective C25-hydroxylation of vitamin D by fungal peroxygenases

Postprint (published version

Towards industrially-feasible delignification and pitch removal by treating paper pulp with Myceliophthora thermophila laccase and a phenolic mediator

The ability of two natural phenols to act as mediators of the recombinant Myceliophthora thermophila laccase (MtL) in eucalypt-pulp delignification was investigated. After alkaline peroxide extraction, the properties of the enzymatically-treated pulps improved with respect to the control. The pulp brightness increased (3.1 points) after the enzymatic treatment with MtL alone, but the highest improvements were obtained after the MtL treatment using syringaldehyde (4.7 points) and especially methyl syringate (8.3 points) as mediators. Likewise, a decrease in kappa number up to 2.7 points was obtained after the MtL–methyl syringate treatment, followed by decreases of 1.4 and 0.9 points after the treatments with MtL–syringaldehyde and MtL alone, respectively. On the other hand, removal of the main lipophilic extractives present in eucalypt pulp was observed after the above laccase–mediator treatments. Finally, the doses of both MtL and methyl syringate were reduced, and results compatible with industrial implementation were obtained.This study was funded by the BIORENEW and LIGNODECO EU projects (NMP2-CT-2006-026456 and KBBE-2009-3-244362, respectively) and the ELLE and RAPERO Spanish MICINN (co-financed by FEDER funds) projects AGL2008-00709 and BIO2008-01533, respectively).Peer reviewe

Molecular determinants for selective C 25-hydroxylation of vitamins D 2 and D 3 by fungal peroxygenases

Hydroxylation of vitamin D by Agrocybe aegerita and Coprinopsis cinerea peroxygenases was investigated in a combined experimental and computational study. 25-Monohydroxylated vitamin D3 (cholecalciferol) and D2 (ergocalciferol), compounds of high interest in human health and animal feeding, can be obtained through reaction with both fungal enzymes. Differences in conversion rates and regioselectivity were nevertheless observed, and, to rationalize the results, diffusion of D2 and D3 on the molecular structure of the two enzymes was performed with PELE software. In good agreement with experimental conversion yields, simulations indicate more favorable energy profiles for the substrates’ entrance in C. cinerea than for A. aegerita enzyme. Furthermore, GC-MS analyses show that while a full regioselective conversion into the active C25 form is catalyzed by C. cinerea peroxygenase for D2 and D3, A. aegerita yielded a mixture of the hydroxylated D3 products. From the molecular simulations, relative distance distributions between the haem compound I oxygen and H24/H25 atoms (hydrogens on C24 and C25 respectively) were plotted. Results show large populations for O-H25 distances below 3 Å for D2 and D3 in C. cinerea in accord with the high reactivity observed for this enzyme. In A. aegerita, however, cholecalciferol has similar populations (below 3 Å) for O-H25 and O-H24 which can justify the small degree of 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-H24 distances below 3 Å and thus, as expected, reaction was not observed in this position.This work was supported by the INDOX (KBBE-2013-7-613549) and PELE (ERC-2009-Adg 25027) EU projects, and by the BIO2011-26694 and CTQ2013-48287 projects of the Spanish Ministry of Economy and Competitiveness.Peer ReviewedPostprint (author's final draft

Oxyfunctionalization of aliphatic compounds by a recombinant peroxygenase from coprinopsis cinerea

The goal of this study is the selective oxyfunctionalization of aliphatic compounds under mild and environmentally friendly conditions using a low-cost enzymatic biocatalyst. This could be possible taking advantage from a new peroxidase type that catalyzes monooxygenase reactions with H2O2 as the only cosubstrate (peroxygenase). With this purpose, recombinant peroxygenase, from gene mining in the sequenced genome of Coprinopsis cinerea and heterologous expression using an industrial fungal host, is tested for the first time on aliphatic substrates. The reaction on free and esterified fatty acids and alcohols, and long-chain alkanes was followed by gas chromatography, and the different reaction products were identified by mass spectrometry. Regioselective hydroxylation of saturated/unsaturated fatty acids was observed at the w-1 and w-2 positions (only at the w-2 position in myristoleic acid). Alkyl esters of fatty acids and monoglycerides were also w-1 or w-2 hydroxylated, but di- and tri-glycerides were not modified. Fatty alcohols yielded hydroxy derivatives at the w-1 or w-2 positions (diols) but also fatty acids and their hydroxy derivatives. Interestingly, the peroxygenase was able to oxyfunctionalize alkanes giving, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule. The predominance of mono- or di-hydroxylated derivatives seems related to the higher or lower proportion of acetone, respectively, in the reaction medium. The recombinant C. cinerea peroxygenase appears as a promising biocatalyst for alkane activation and production of aliphatic oxygenated derivatives, with better properties than the previously reported peroxygenase from Agrocybe aegerita, and advantages related to its recombinant nature for enzyme engineering and industrial production.This study was funded by the PEROXICATS (KBBE-2010-4-265397) EU project. E.D. Babot thanks the Spanish CSIC for a JAE fellowship co-financed by FSE. R. Ullrich and M. Hofrichter are acknowledged for providing wild-type A. aegerita peroxygenase preparation.Peer reviewe

Steroid hydroxylation by basidiomycete peroxygenases: A combined experimental and computational study, Applied and environmental microbiology

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.This study was supported by the INDOX (KBBE-2013-7-613549), PEROXICATS (KBBE-2010-4-265397), and PELE (ERC-2009-Adg 25027) EU projects.Peer Reviewe

Steroid hydroxylation by basidiomycete peroxygenases: A combined experimental and computational study, Applied and environmental microbiology

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.This study was supported by the INDOX (KBBE-2013-7-613549), PEROXICATS (KBBE-2010-4-265397), and PELE (ERC-2009-Adg 25027) EU projects.Peer Reviewe