BUSCADOR RECOLECTA

Background: Pleurotus ostreatus is the second edible mushroom worldwide, and a model fungus for delignification
applications, with the advantage of growing on woody and nonwoody feedstocks. Its sequenced genome is available,
and this gave us the opportunity to perform proteomic studies to identify the enzymes overproduced in lignocellulose
cultures.
Results: Monokaryotic P. ostreatus (PC9) was grown with poplar wood or wheat straw as the sole C/N source and the
extracellular proteins were analyzed, together with those from glucose medium. Using nano-liquid chromatography
coupled to tandem mass spectrometry of whole-protein hydrolyzate, over five-hundred proteins were identified.
Thirty-four percent were unique of the straw cultures, while only 15 and 6 % were unique of the glucose and poplar
cultures, respectively (20 % were produced under the three conditions, and additional 19 % were shared by the two
lignocellulose cultures). Semi-quantitative analysis showed oxidoreductases as the main protein type both in the
poplar (39 % total abundance) and straw (31 %) secretomes, while carbohydrate-active enzymes (CAZys) were only
slightly overproduced (14–16 %). Laccase 10 (LACC10) was the main protein in the two lignocellulose secretomes
(10–14 %) and, together with LACC2, LACC9, LACC6, versatile peroxidase 1 (VP1), and manganese peroxidase 3
(MnP3), were strongly overproduced in the lignocellulose cultures. Seven CAZys were also among the top-50 proteins,
but only CE16 acetylesterase was overproduced on lignocellulose. When the woody and nonwoody secretomes
were compared, GH1 and GH3 β-glycosidases were more abundant on poplar and straw, respectively and, among less
abundant proteins, VP2 was overproduced on straw, while VP3 was only found on poplar. The treated lignocellulosic
substrates were analyzed by two-dimensional nuclear magnetic resonance (2D NMR), and a decrease of lignin relative
to carbohydrate signals was observed, together with the disappearance of some minor lignin substructures, and an
increase of sugar reducing ends.
Conclusions: Oxidoreductases are strongly induced when P. ostreatus grows on woody and nonwoody lignocellulosic
substrates. One laccase occupied the first position in both secretomes, and three more were overproduced
together with one VP and one MnP, suggesting an important role in lignocellulose degradation. Preferential removal
of lignin vs carbohydrates was shown by 2D NMR, in agreement with the above secretomic results., This work was supported by the INDOX (KBBE-2013-613549) EU project, the BIO2014-56388-R, AGL2014-53730-R, and AGL2011-30495 projects of the Spanish Ministry of Economy and Competitiveness (MINECO) co-financed by FEDER funds, and the ProteoRed platform of the Spanish Institute of Health Carlos III (ISCIII). The work conducted by the US DOE JGI is supported by the Office of Science of the US DOE under contract number DE-AC02-05CH11231. FJR-D thanks a Ramón y Cajal contract of the Spanish MINECO, and JR thanks a contract of the CSIC project 201440E097.

Aryl-alcohol oxidase (AAO) has demonstrated to be an enzyme with a bright future ahead due to its biotechnological potential in deracemisation of chiral compounds, production of bioplastic precursors and other reactions of interest. Expanding our understanding on the AAO reaction mechanisms, through the investigation of its structure-function relationships, is crucial for its exploitation as an industrial biocatalyst. In this regard, previous computational studies suggested an active role for AAO Phe397 at the active-site entrance. This residue is located in a loop that partially covers the access to the cofactor forming a bottleneck together with two other aromatic residues. Kinetic and affinity spectroscopic studies, complemented with computational simulations using the recently developed adaptive-PELE technology, reveal that the Phe397 residue is important for product release and to help the substrates attain a catalytically relevant position within the active-site cavity. Moreover, removal of aromaticity at the 397 position impairs the oxygen-reduction activity of the enzyme. Experimental and computational findings agree very well in the timing of product release from AAO, and the simulations help to understand the experimental results. This highlights the potential of adaptive-PELE to provide answers to the questions raised by the empirical results in the study of enzyme mechanisms., This work was supported by the EnzOx2 project (H2020-BBI-PPP-2015-720297) of the European Joint Undertaking of Bio-based Industries (http://bbi-europe.eu), the INDOX project (KBBE-2013-7-613549) of the
European Seventh Framework Programme, and the NOESIS (BIO2014-56388-R), vMutate (CTQ2016-79138-R)
and FLADIMOTEC (BIO2016-75183-P) projects of the Spanish Ministry of Economy and Competitiveness. J.C. acknowledges a FPU fellowship (FPU2012-2041) from the Spanish Ministry of Education, Culture and Sports., Peer Reviewed

Due to its importance in the pharmaceutical industry, ligand dynamic simulations have experienced a great expansion. Using all-atom models and cutting edge hardware, one can perform non-biased ligand migration, active site search and binding studies. In this letter we demonstrate (and validate by PCR mutagenesis) how these techniques, when combined with quantum mechanics, open new possibilities in enzyme engineering. We provide a complete analysis where: 1) biophysical simulations produce ligand diffusion and, 2) biochemical modeling samples the chemical event. Using such broad analysis we engineer a highly stable peroxidase activating the enzyme for new substrate
oxidation after rational mutation of two non-conserved surface residues. In particular, we create a new surface-binding site, quantitatively predicting the in vitro change in oxidation rate obtained by mutagenic PCR and achieving a comparable specificity constant to active peroxidases., This work was supported by the INDOX (KBBE-2013-7-613549 to ATM) European project, and the CTQ2013-48287 (to VG) and BIO2014-56388-R (to FJR-D) projects of the Spanish Ministry of Economy and Competitiveness (MINECO). FJR-D acknowledges a MINECO
Ramón&Cajal contract., Peer Reviewed

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., This work has been funded by the INDOX European project (KBBE-2013-7-613549), together with the BIO2014-56388-R and AGL2014-53730-R projects of the Spanish Ministry of Economy and Competitiveness (MINECO) co-financed by FEDER funds, and the BBI JU project EnzOx2 (H2020-BBI-PPP-2015-2-720297). The work conducted
by the US DOE JGI was supported by the Office of Science of the US DOE under contract number DE-AC02-05CH11231. The authors thank other members of their groups at CIB-CSIC, Novozymes, Technical University of Dresden, JenaBios, University of Naples
Federico II, Setas Kimya Sanayy, Wageningen University & Research, Anaxomics, Chiracon, BOKU, Delft University of Technology, INRABBF, Biopolis, Cheminova, CLEA, Solvay, IRNAS-CSIC and ICP-CSIC for their significant contributions to the results presented., Peer Reviewed

A dye-decolorizing peroxidase (DyP) from Pleurotus ostreatus (PosDyP4) catalyzes the oxidation of Mn2+ to Mn3+, in the presence of H2O2, with an efficiency similar to the well-known manganese peroxidases and versatile peroxidases from this and other white-rot fungi. PosDyP4 has been overexpressed in Escherichia coli as an active enzyme, and its crystal structure has been solved at 1.56 Å resolution. A combination of substrate diffusion simulations on the solved structure using the PELE software, electron paramagnetic resonance, and site-directed mutagenesis led to identification of the residues involved in Mn2+ oxidation. The oxidation site in PosDyP4 is different than the conserved site in the other Mn-oxidizing peroxidases mentioned above, and it includes four acidic residues (three aspartates and one glutamate) located at the surface of the protein. Moreover, since the Mn2+ ion is not in direct contact with the heme propionates, a tyrosine residue participates in the electron transfer to the cofactor, being the only essential individual residue for PosDyP4 oxidation of the metal ion. The four acidic residues contribute to Mn2+ binding in different extents, with the glutamate also involved in the initial electron transfer to the key tyrosine, as confirmed by the >50-fold decreased kcat after removing its side-chain carboxylic group. A second electron transfer pathway operates in PosDyP4 for the oxidation of aromatics and dyes starting at a surface tryptophan, as reported in other fungal and prokaryotic DyPs, and connecting with the final part of the Mn2+ oxidation route. Both tryptophanyl and tyrosyl radicals, potentially involved in catalysis, were detected by electron paramagnetic resonance of the native enzyme and its tryptophan-less variant, respectively., This work has been funded by the H2020 BBI-JU (www.bbi-europe.eu) project EnzOx2 (BBI-PPP-2015-2-720297; www. enzox2.eu) and the FP7 project INDOX (KBBE-2013-7-613549;
www.indoxproject.eu), together with the BIO2014-56388-R (NOESIS), BFU2016-77835-R, CTQ2016-79138-R,
and (BIO2017-86559-R (GENOBIOREF) projects of the Spanish Ministry of Economy, Industry and Competitiveness, cofinanced by FEDER funds. The sequencing of P. ostreatus
genome at JGI was supported by the DOE Office of Science, under Contract No. DE-AC02-05CH11231. We thank the staff
of the ID30B beamline at ESRF (Grenoble, France) for assistance. CSGI (Florence, Italy) FFABR 2017 (Legge 232/ 2016 Comma 295, and Legge di Bilancio 2017) and MIUR
(Dipartimento di Eccellenza, 2018-2022) grants are acknowledged for financial support., Peer Reviewed

Iterative saturation mutagenesis was performed over six residues delimiting the substrate binding pocket of a high redox potential chimeric laccase with the aim of enhancing its activity over sinapic acid, a ligninrelated phenol of industrial interest. In total, more than 15000 clones were screened and two selected variants, together with the parent-type laccase, were purified and characterized. The new variants presented shifted pH activity profiles and enhanced turnover rates on sinapic acid and its methyl ester, whereas the oxidation of related phenols was not significantly enhanced. Neither the enzyme's redox potential nor the mechanism of the reaction was affected. Quantum mechanics and molecular dynamics calculations were done to rationalize the effect of the selected mutations, revealing the critical role of the residues of the enzyme pocket to provide the precise binding of the substrate that enables an efficient electron transfer to the T1 copper. The results presented highlight the power of combining directed evolution and computational approaches to give novel solutions in enzyme engineering and to understand the mechanistic reasons behind them, offering new insights for further rational design towards specific targets., This work was funded by INDOX (KBBE-2013-7-613549) European project and NOESIS (BIO2014-56388-R) and CTQ2013-
48287-R Spanish National Projects. I. P. and G. S. acknowledge the Spanish Research Council (CSIC) and MINECO for their respective predoctoral fellowships., Peer Reviewed

Combining a computational analysis with site-directed mutagenesis, we have studied the long-range electron transfer pathway in versatile and lignin peroxidases, two enzymes of biotechnological interest that play a key role for fungal degradation of the bulky lignin molecule in plant biomass. The in silico study established two possible electron transfer routes starting at the surface tryptophan residue previously identified as responsible for oxidation of the bulky lignin polymer. Moreover, in both enzymes, a second buried tryptophan residue appears as a top electron transfer carrier, indicating the prevalence of one pathway. Site-directed mutagenesis of versatile peroxidase (from Pleurotus eryngii) allowed us to corroborate the computational analysis and the role played by the buried tryptophan (Trp244) and a neighbor phenylalanine residue (Phe198), together with the surface tryptophan, in the electron transfer. These three aromatic residues are highly conserved in all the sequences analyzed (up to a total of 169). The importance of the surface (Trp171) and buried (Trp251) tryptophan residues in lignin peroxidase has been also confirmed by directed mutagenesis of the Phanerochaete chrysosporium enzyme. Overall, the combined procedure identifies analogous electron transfer pathways in the long-range oxidation mechanism for both ligninolytic peroxidases, constituting a good example of how computational analysis avoids making extensive trial-error mutagenic experiments., This work was supported by the INDOX (KBBE-2013-7-613549) EU project, and by the projects EnzOx2 (H2020-BBI-PPP-2015-2-720297) of the Joint Undertaking of European BioBased Industries (www.bbi-europe.eu), and BIO2014-56388-R (NOESIS) and CTQ2016-79138-R of the SpanishMinistry of Economy and Competitiveness (MINECO). F.J.R.-D. acknowledges a MINECO Ramón & Cajal contract., Peer Reviewed

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 Reviewed

To meet the very specific requirements demanded by industry, proteins must be appropriately tailored. Engineering laccases, to improve the oxidation of small molecules, with applications in multiple fields, is, however, a difficult task. Most efforts have concentrated on increasing the redox potential of the enzyme, but in recent work, we have pursued an alternate strategy to engineering these biocatalysts. In particular, we have found that redesigning substrate binding at the T1 pocket, guided by in silico methodologies, to be a more consistent option. In this work, we evaluate the robustness of our computational approach to estimate activity, emphasizing the importance of the binding event in laccase reactivity. Strengths and weaknesses of the protocol are discussed along with its potential for scoring large numbers of protein sequences and thus its significance in protein engineering., This study was supported by the OxiDesign (CTQ2013-48287-R) and NOESIS (BIO2014-56388-R) Spanish projects and the
INDOX (KBBE-2013-7−613549) EU-project., Peer Reviewed

Aryl-alcohol oxidase (AAO) has demonstrated to be an enzyme with a bright future ahead due to its biotechnological potential in deracemisation of chiral compounds, production of bioplastic precursors and other reactions of interest. Expanding our understanding on the AAO reaction mechanisms, through the investigation of its structure-function relationships, is crucial for its exploitation as an industrial biocatalyst. In this regard, previous computational studies suggested an active role for AAO Phe397 at the active-site entrance. This residue is located in a loop that partially covers the access to the cofactor forming a bottleneck together with two other aromatic residues. Kinetic and a nity spectroscopic studies, complemented with computational simulations using the recently developed adaptive-PELE technology, reveal that the Phe397 residue is important for product release and to help the substrates attain a catalytically relevant position within the active-site cavity. Moreover, removal of aromaticity at the 397 position impairs the oxygen-reduction activity of the enzyme. Experimental and computational ndings agree very well in the timing of product release from AAO, and the simulations help to understand the experimental results. This highlights the potential of adaptive-PELE to provide answers to the questions raised by the empirical results in the study of enzyme mechanisms.

The temperature dependence of hydride transfer from the substrate to the N5 of the FAD cofactor during the reductive half-reaction of Pleurotus eryngii aryl-alcohol oxidase (AAO) is assessed here. Kinetic isotope effects on both the pre-steady state reduction of the enzyme and its steady-state kinetics, with differently deuterated substrates, suggest an environmentally-coupled quantum-mechanical tunnelling process. Moreover, those kinetic data, along with the crystallographic structure of the enzyme in complex with a substrate analogue, indicate that AAO shows a pre-organized active site that would only require the approaching of the hydride donor and acceptor for the tunnelled transfer to take place. Modification of the enzyme''s active-site architecture by replacement of Tyr92, a residue establishing hydrophobic interactions with the substrate analogue in the crystal structure, in the Y92F, Y92L and Y92W variants resulted in different temperature dependence patterns that indicated a role of this residue in modulating the transfer reaction.

The mechanism of dioxygen reduction by the flavoenzyme aryl-alcohol oxidase was investigated with kinetic isotope, viscosity, and pL (pH/pD) effects in rapid kinetics experiments by stopped-flow spectrophotometry of the oxidative half-reaction of the enzyme. Double mixing of the enzyme in a stopped-flow spectrophotometer with [a-2H2]-p-methoxybenzyl alcohol and oxygen at varying aging times established a slow rate constant of 0.0023 s-1 for the wash-out of the D atom from the N5 atom of the reduced flavin. Thus, the deuterated substrate could be used to probe the cleavage of the N-H bond of the reduced flavin in the oxidative half-reaction. A significant and pH-independent substrate kinetic isotope effect (KIE) of 1.5 between pH 5.0 and 8.0 demonstrated that H transfer is partially limiting the oxidative half-reaction of the enzyme; a negligible solvent KIE of 1.0 between pD 5.0 and 8.0 proved a fast H+ transfer reaction that does not contribute to determining the flavin oxidation rates. Thus, a mechanism for dioxygen reduction in which the H atom originating from the reduced flavin and a H+ from a solvent exchangeable site are transferred in separate kinetic steps is proposed. The spectroscopic and kinetic data presented also showed a lack of stabilization of transient flavin intermediates. The substantial differences in the mechanistic details of O2 reduction by aryl-alcohol oxidase with respect to other alcohol oxidases like choline oxidase, pyranose 2-oxidase, and glucose oxidase further demonstrate the high level of versatility of the flavin cofactor in flavoenzymes.