Asymmetric sulfoxidation by engineering the heme pocket of a dye-decolorizing peroxidase

The so-called dye-decolorizing peroxidases (DyPs) constitute a new family of proteins exhibiting remarkable stability. With the aim of providing them new catalytic activities of biotechnological interest, the heme pocket of one of the few DyPs fully characterized to date (from the fungus Auricularia auricula-judae) was redesigned based on the crystal structure available, and its potential for asymmetric sulfoxidation was evaluated. Chiral sulfoxides are important targets in organic synthesis and enzyme catalysis, due to a variety of applications. Interestingly, one of the DyP variants, F359G, is highly stereoselective in sulfoxidizing methylphenyl sulfide and methyl-p-tolyl sulfide (95–99% conversion, with up to 99% excess of the S enantiomer in short reaction times), while the parent DyP has no sulfoxidation activity, and the L357G variant produces both R and S enantiomers. The two variants were crystallized, and their crystal structures were used in molecular simulations to provide a rational explanation for the new catalytic activities. Protein energy landscape exploration (PELE) showed more favorable protein–substrate catalytic complexes for the above variants, with a considerable number of structures near the oxygen atom of the activated heme, which is incorporated into the substrates as shown in 18O-labeling experiments, and improved affinity with respect to the parent enzyme, explaining their sulfoxidation activity. Additional quantum mechanics/molecular mechanics (QM/MM) calculations were performed to elucidate the high stereoselectivity observed for the F359G variant, which correlated with higher reactivity on the substrate molecules adopting pro-S poses at the active site. Similar computational analyses can help introduce/improve (stereoselective) sulfoxidation activity in related hemeproteins.This work was supported by the INDOX (KBBE-2013-7-613549) EU project and by the BIO2014-56388-R (NOESIS), BFU2014-55448-P and CTQ2013-48287-R projects of the Spanish Ministry of Economy and Competitiveness (MINECO). Pedro Merino (University of Zaragoza, Spain) is acknowledged for his suggestions on chiral HPLC analyses, and Alicia Prieto and Leonor Rodríguez (CIB, Madrid, Spain) for their help in GC-MS analyses. We cordially thank the staff at ID23-1 beamline (ESRF) and the BL13-XALOC beamline (ALBA). F. J. R.-D. acknowledges a MINECO Ramón & Cajal contract.Peer ReviewedPostprint (published version

A Functional Role of RB-Dependent Pathway in the Control of Quiescence in Adult Epidermal Stem Cells Revealed by Genomic Profiling

Continuous cell renewal in mouse epidermis is at the expense of a pool of pluripotent cells that lie in a well defined niche in the hair follicle known as the bulge. To identify mechanisms controlling hair follicle stem cell homeostasis, we developed a strategy to isolate adult bulge stem cells in mice and to define their transcriptional profile. We observed that a large number of transcripts are underexpressed in hair follicle stem cells when compared to non-stem cells. Importantly, the majority of these downregulated genes are involved in cell cycle. Using bioinformatics tools, we identified the E2F transcription factor family as a potential element involved in the regulation of these transcripts. To determine their functional role, we used engineered mice lacking Rb gene in epidermis, which showed increased expression of most E2F family members and increased E2F transcriptional activity. Experiments designed to analyze epidermal stem cell functionality (i.e.: hair regrowth and wound healing) imply a role of the Rb-E2F axis in the control of stem cell quiescence in epidermis

Continuous fungal treatment of non-sterile veterinary hospital effluent: pharmaceuticals removal and microbial community assessment

Source point treatment of effluents with a high load of pharmaceutical active compounds (PhACs), such as hospital wastewater, is a matter of discussion among the scientific community. Fungal treatments have been reported to be successful in degrading this type of pollutants and, therefore, the white-rot fungus Trametes versicolor was applied for the removal of PhACs from veterinary hospital wastewater. Sixty-six percent removal was achieved in a non-sterile batch bioreactor inoculated with T. versicolor pellets. On the other hand, the study of microbial communities by means of DGGE and phylogenetic analyses led us to identify some microbial interactions and helped us moving to a continuous process. PhAC removal efficiency achieved in the fungal treatment operated in non-sterile continuous mode was 44 % after adjusting the C/N ratio with respect to the previously calculated one for sterile treatments. Fungal and bacterial communities in the continuous bioreactors were monitored as well.Authors want to acknowledge the UAB veterinary hospital staff for their kind permission and help for the samplings. This work has been funded by the Spanish Ministry of Economy and Competitiveness and FEDER (projects CTM2013-48545-C2 and AIB2010PT-00169) and supported by the Generalitat de Catalunya (Consolidated Research Groups 2014-SGR-476 and 2014-SGR-291). The Department of Chemical Engineering of the Universitat Autonoma de Barcelona (UAB) is a member of the Xarxa de Referencia en Biotecnologia de la Generalitat de Catalunya. M. Badia-Fabregat and D. Lucas acknowledge the predoctoral grants from UAB and from the Spanish Ministry of Education, Culture and Sports (AP-2010-4926), respectively. The authors also thank the Portuguese Foundation for Science and Technology (FCT) Strategic Project PEst-OE/EQB/LA0023/2013, Project FCOMP-01-0124-FEDER-027462 co-funded by Operational Competitiveness Programme, FEDER, and Project "BioEnv-Biotechnology and Bioengineering for a sustainable world," REF. NORTE-07-0124-FEDER-000048, co-funded by Programa Operacional Regional do Norte (ON.2 - O Novo Norte), QREN, FEDER

Asymmetric sulfoxidation by engineering the heme pocket of a dye-decolorizing peroxidase

The so-called dye-decolorizing peroxidases (DyPs) constitute a new family of proteins exhibiting remarkable stability. With the aim of providing them new catalytic activities of biotechnological interest, the heme pocket of one of the few DyPs fully characterized to date (from the fungus Auricularia auricula-judae) was redesigned based on the crystal structure available, and its potential for asymmetric sulfoxidation was evaluated. Chiral sulfoxides are important targets in organic synthesis and enzyme catalysis, due to a variety of applications. Interestingly, one of the DyP variants, F359G, is highly stereoselective in sulfoxidizing methylphenyl sulfide and methyl-p-tolyl sulfide (95–99% conversion, with up to 99% excess of the S enantiomer in short reaction times), while the parent DyP has no sulfoxidation activity, and the L357G variant produces both R and S enantiomers. The two variants were crystallized, and their crystal structures were used in molecular simulations to provide a rational explanation for the new catalytic activities. Protein energy landscape exploration (PELE) showed more favorable protein–substrate catalytic complexes for the above variants, with a considerable number of structures near the oxygen atom of the activated heme, which is incorporated into the substrates as shown in 18O-labeling experiments, and improved affinity with respect to the parent enzyme, explaining their sulfoxidation activity. Additional quantum mechanics/molecular mechanics (QM/MM) calculations were performed to elucidate the high stereoselectivity observed for the F359G variant, which correlated with higher reactivity on the substrate molecules adopting pro-S poses at the active site. Similar computational analyses can help introduce/improve (stereoselective) sulfoxidation activity in related hemeproteins.This work was supported by the INDOX (KBBE-2013-7-613549) EU project and by the BIO2014-56388-R (NOESIS), BFU2014-55448-P and CTQ2013-48287-R projects of the Spanish Ministry of Economy and Competitiveness (MINECO). Pedro Merino (University of Zaragoza, Spain) is acknowledged for his suggestions on chiral HPLC analyses, and Alicia Prieto and Leonor Rodríguez (CIB, Madrid, Spain) for their help in GC-MS analyses. We cordially thank the staff at ID23-1 beamline (ESRF) and the BL13-XALOC beamline (ALBA). F. J. R.-D. acknowledges a MINECO Ramón & Cajal contract.Peer Reviewe

Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and site-directed mutagenesis study

Dye-decolorizing peroxidase (DyP) of Auricularia auricula-judae has been expressed in E. coli, as a representative for new DyP family, and subjected to mutagenesis, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE software provided several binding-energy optima for the anthraquinone-type Reactive Blue 19 (RB19) near the above aromatic residues, and the haem access-channel. Subsequent QM/MM calculations showed a higher tendency of Trp-377 than other exposed haem-neighbour residues to harbour a catalytic protein radical, and identified the electrontransfer pathway. The existence of such a radical in H2O2-activated DyP was shown by low temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high and low turnover sites. The high-turnover site for oxidation of RB19 (kcat> 200 s-1) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19 kcat ~20 s-1) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant

Electrogenic ion transport in cultured human sweat gland epithelia Control and cystic fibrosis

SIGLEAvailable from British Library Document Supply Centre- DSC:D60655 / BLDSC - British Library Document Supply CentreGBUnited Kingdo

Catalytic surface radical in dye-decolorizing peroxidase: A computational, spectroscopic and directed mutagenesis study

Dye-decolorizing peroxidase (DyP) of Auricularia auriculajudae has been expressed in Escherichia coli as a representative of a new DyP family, and subjected to mutagenic, spectroscopic, crystallographic and computational studies. The crystal structure of DyP shows a buried haem cofactor, and surface tryptophan and tyrosine residues potentially involved in long-range electron transfer from bulky dyes. Simulations using PELE (Protein Energy Landscape Exploration) software provided several binding-energy optima for the anthraquinone-type RB19 (Reactive Blue 19) near the above aromatic residues and the haem access-channel. Subsequent QM/MM (quantum mechanics/molecular mechanics) calculations showed a higher tendency of Trp-377 than other exposed haem-neighbouring residues to harbour a catalytic protein radical, and identified the electron-transfer pathway. The existence of such a radical in H2O2 activated DyP was shown by low-temperature EPR, being identified as a mixed tryptophanyl/tyrosyl radical in multifrequency experiments. The signal was dominated by the Trp-377 neutral radical contribution, which disappeared in the W377S variant, and included a tyrosyl contribution assigned to Tyr-337 after analysing the W377S spectra. Kinetics of substrate oxidation by DyP suggests the existence of high- and low-turnover sites. The high-turnover site for oxidation of RB19 (kcat>200 s−1) and other DyP substrates was assigned to Trp-377 since it was absent from the W377S variant. The low-turnover site/s (RB19kcat∼20 s−1) could correspond to the haem access-channel, since activity was decreased when the haem channel was occluded by the G169L mutation. If a tyrosine residue is also involved, it will be different from Tyr-337 since all activities are largely unaffected in the Y337S variant.We thank the staff of the SOLEIL (Gyf-sur-Yvette, France) and ALBA (Barcelona, Spain) synchrotrons, and the BSC (Barcelona, Spain) computational facilities. The MALDI– TOF analyses were carried out at the CIB Proteomics facility, a member of the Spanish ProteoRed-ISCIII network.This work was supported by the INDOX [grant number KBBE-2013-7-613549] and PELE [grant number ERC-2009-Adg 25027] European Union projects, by projects of the Spanish Ministry of Economy and Competitiveness (MINECO) [grant number BIO2011-26694, CTQ2013-48287 and BFU2011-24615] and by the Italian Ministry of Education, Universities and Research (MIUR) [project PRIN 2009-STNWX3]. D.L. and F.J.R.-D. are grateful for the financial support of an EU project contract, and a Ramon y Cajal contract of the Spanish Ministry of Economy and Competitiveness (MINECO) respectively.Peer Reviewe