BUSCADOR RECOLECTA

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 Reviewed

This is the peer reviewed version of the following article, which has been published in final form at 10.1002/cbic.201500493. This article may be used for non-commercial purposes in accordance With Wiley-VCH Terms and Conditions for self-archiving, There is an increasing interest in enzymes that catalyze the hydroxylation of naphthalene under mild conditions and with minimal requirements. To address this challenge, an extracellular fungal aromatic peroxygenase with mono(per)oxygenase activity was engineered to convert naphthalene selectively into 1-naphthol. Mutant libraries constructed by random mutagenesis and DNA recombination were screened for peroxygenase activity on naphthalene together with quenching of the undesired peroxidative activity on 1-naphthol (one-electron oxidation). The resulting double mutant (G241D-R257K) obtained from this process was characterized biochemically and computationally. The conformational changes produced by directed evolution improved the substrate's catalytic position. Powered exclusively by catalytic concentrations of H2O2, this soluble and stable biocatalyst has a total turnover number of 50 000, with high regioselectivity (97 %) and reduced peroxidative activity., We thank Paloma Santos Moriano (ICP, CSIC, Spain) for assistance with the HPLC and LC/MS analysis, and Jesper Vind (Novozymes, Denmark) and Angel T. Martinez (CIB, CSIC, Spain) for helpful discussions. This work was supported by the European Commission projects Indox-FP7-KBBE-2013-7-613549 and Cost-Action CM1303-Systems Biocatalysis, and the National Projects Dewry [BIO201343407-R], Cambios [RTC-2014-1777-3] and OXYdesign [CTQ2013-48287-R]., Peer Reviewed

This is the peer reviewed version of the following article: [Ferreira, P., Hernández-Ortega, A., Lucas, F., Carro, J., Herguedas, B., Borrelli, K. W., Guallar, V., Martínez, A. T. and Medina, M. (2015), Aromatic stacking interactions govern catalysis in aryl-alcohol oxidase. FEBS J, 282: 3091–3106. doi:10.1111/febs.13221], which has been published in final form at [10.1111/febs.13221]. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving." http://olabout.wiley.com/WileyCDA/Section/id-820227.html
The version posted may not be updated or replaced with the final published version (the Version of Record)., Aryl-alcohol oxidase (AAO, EC 1.1.3.7) generates H2O2 for lignin degradation at the expense of benzylic and other π system-containing primary alcohols, which are oxidized to the corresponding aldehydes. Ligand diffusion studies on Pleurotus eryngii AAO showed a T-shaped stacking interaction between the Tyr92 side chain and the alcohol substrate at the catalytically competent position for concerted hydride and proton transfers. Bi-substrate kinetics analysis revealed that reactions with 3-chloro- or 3-fluorobenzyl alcohols (halogen substituents) proceed via a ping–pong mechanism. However, mono- and dimethoxylated substituents (in 4-methoxybenzyl and 3,4-dimethoxybenzyl alcohols) altered the mechanism and a ternary complex was formed. Electron-withdrawing substituents resulted in lower quantum mechanics stacking energies between aldehyde and the tyrosine side chain, contributing to product release, in agreement with the ping–pong mechanism observed in 3-chloro- and 3-fluorobenzyl alcohol kinetics analysis. In contrast, the higher stacking energies when electron donor substituents are present result in reaction of O2 with the flavin through a ternary complex, in agreement with the kinetics of methoxylated alcohols. The contribution of Tyr92 to the AAO reaction mechanism was investigated by calculation of stacking interaction energies and site-directed mutagenesis. Replacement of Tyr92 by phenylalanine does not alter the AAO kinetic constants (on 4-methoxybenzyl alcohol), most probably because the stacking interaction is still possible. However, introduction of a tryptophan residue at this position strongly reduced the affinity for the substrate (i.e. the pre-steady state Kd and steady-state Km increase by 150-fold and 75-fold, respectively), and therefore the steady-state catalytic efficiency, suggesting that proper stacking is impossible with this bulky residue. The above results confirm the role of Tyr92 in substrate binding, thus governing the kinetic mechanism in AAO., This work was supported by the BIO2013-42978-P (to MM), BIO2011-26694 (to ATM), “Juan de la Cierva” (to FL) and CTQ2010-18123 (to VG) Grants of the Spanish Ministry of Economy and Competitiveness (MINECO) and by the INDOX (KBBE-2013-7-613549, to ATM) and PELE (ERC-2009-Adg 25027, to VG) European projects., Peer Reviewed

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 Reviewed

Ligninolytic peroxidases are enzymes of biotechnological interest due to their ability to oxidize high redox potential aromatic compounds, including the recalcitrant lignin polymer. However, different obstacles prevent their use in industrial and environmental applications, including low stability towards their natural oxidizing-substrate H2O2. In this work, versatile peroxidase was taken as a model ligninolytic peroxidase, its oxidative inactivation by H2O2 was studied and different strategies were evaluated with the aim of improving H2O2 stability.
Oxidation of the methionine residues was produced during enzyme inactivation by H2O2 excess. Substitution of these residues, located near the heme cofactor and the catalytic tryptophan, rendered a variant with a 7.8-fold decreased oxidative inactivation rate. A second strategy consisted in mutating two residues (Thr45 and Ile103) near the catalytic distal histidine with the aim of modifying the reactivity of the enzyme with H2O2. The T45A/I103T variant showed a 2.9-fold slower reaction rate with H2O2 and 2.8-fold enhanced oxidative stability. Finally, both strategies were combined in the T45A/I103T/M152F/M262F/M265L variant, whose stability in the presence of H2O2 was improved 11.7-fold. This variant showed an increased half-life, over 30 min compared with 3.4 min of the native enzyme, under an excess of 2000 equivalents of H2O2. Interestingly, the stability improvement achieved was related with slower formation, subsequent stabilization and slower bleaching of the enzyme Compound III, a peroxidase intermediate that is not part of the catalytic cycle and leads to the inactivation of the enzyme., This work was funded by the Commission
of the European Communities through the INDOX project (KBBE-2013-7-613549, "Optimized oxidoreductases for medium and large scale industrial biotransformations"), and by the Spanish Ministerio de Economía y Competitividad (MINECO) through the HIPOP project (BIO2011-26694, “Screening and engineering of new high-redoxpotential peroxidases”). VS-J and FJR-D thank the
financial support of a research fellowship (Formación de Personal Investigador, FPI) and a Ramón y Cajal contract of the Spanish MINECO, respectively. The authors have no conflict of interest to declare. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of
the manuscript., Peer Reviewed

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn2+. Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

Oxidative conversion of 5-hydroxymethylfurfural (HMF) is of biotechnological interest for the production of renewable (lignocellulose-based) platform chemicals, such as 2,5-furandicarboxylic acid (FDCA). To the best of our knowledge, the ability of fungal aryl-alcohol oxidase (AAO) to oxidize HMF is reported here for the first time, resulting in almost complete conversion into 2,5-formylfurancarboxylic acid (FFCA) in a few hours. The reaction starts with alcohol oxidation, yielding 2,5-diformylfuran (DFF), which is rapidly converted into FFCA by carbonyl oxidation, most probably without leaving the enzyme active site. This agrees with the similar catalytic efficiencies of the enzyme with respect to oxidization of HMF and DFF, and its very low activity on 2,5-hydroxymethylfurancarboxylic acid (which was not detected by GC-MS). However, AAO was found to be unable to directly oxidize the carbonyl group in FFCA, and only modest amounts of FDCA are formed from HMF (most probably by chemical oxidation of FFCA by the H2O2 previously generated by AAO). As aldehyde oxidation by AAO proceeds via the corresponding geminal diols (aldehyde hydrates), the various carbonyl oxidation rates may be related to the low degree of hydration of FFCA compared with DFF. The conversion of HMF was completed by introducing a fungal unspecific heme peroxygenase that uses the H2O2 generated by AAO to transform FFCA into FDCA, albeit more slowly than the previous AAO reactions. By adding this peroxygenase when FFCA production by AAO has been completed, transformation of HMF into FDCA may be achieved in a reaction cascade in which O2 is the only co-substrate required, and water is the only by-product formed.

The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) recently were sequenced to expand our knowledge on the diversity and distribution of genes involved in degradation of plant polymers in this Basidiomycota order, which includes most wood-rotting fungi. Oxidases, including members of the glucose-methanol-choline (GMC) oxidoreductase superfamily, play a central role in the above degradative process because they generate extracellular H2O2 acting as the ultimate oxidizer in both white-rot and brown-rot decay. The survey was completed by analyzing the GMC genes in the available genomes of seven more species to cover the four Polyporales clades. First, an in silico search for sequences encoding members of the aryl-alcohol oxidase, glucose oxidase, methanol oxidase, pyranose oxidase, cellobiose dehydrogenase and pyranose dehydrogenase families was performed. The curated sequences were subjected to an analysis of their evolutionary relationships, followed by estimation of gene duplication/ reduction history during fungal evolution. Second, the molecular structures of the near one hundred GMC oxidoreductases identified were modeled to gain insight into their structural variation and expected catalytic properties. In contrast to ligninolytic peroxidases, whose genes are present in all white-rot Polyporales genomes and absent from those of brown-rot species, the H2O2-generating oxidases are widely distributed in both fungal types. This indicates that the GMC oxidases provide H2O2 for both ligninolytic peroxidase activity (in white-rot decay) and Fenton attack on cellulose (in brown-rot decay), after the transition between both decay patterns in Polyporales occurred.