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The vanadium-dependent chloroperoxidase from Curvularia inaequalis is a stable and efficient biocatalyst for the hydroxyhalogenation of a broad range of alkenes into halohydrins. Up to 1 200 000 TON with 69 s−1 TOF were observed for the biocatalyst. A bienzymatic cascade to yield epoxides as reaction products is presented.

The variable C-terminal tail of manganese peroxidases, a group of enzymes involved in lignin degradation, is implicated in their catalytic and stability properties, as shown by new crystal structures, molecular-simulation and directed-mutagenesis data. Based on this structural–functional evaluation, short and long/extralong manganese peroxidase subfamilies have been accepted; the latter are characterized by exceptional stability, while it is shown for the first time that the former are able to oxidize other substrates at the same site where manganese(II) is oxidized., The genome of Ceriporiopsis subvermispora includes 13 manganese peroxidase (MnP) genes representative of the three subfamilies described in ligninolytic fungi, which share an Mn2+-oxidation site and have varying lengths of the C-terminal tail. Short, long and extralong MnPs were heterologously expressed and biochemically characterized, and the first structure of an extralong MnP was solved. Its C-terminal tail surrounds the haem-propionate access channel, contributing to Mn2+ oxidation by the internal propionate, but prevents the oxidation of 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), which is only oxidized by short MnPs and by shortened-tail variants from site-directed mutagenesis. The tail, which is anchored by numerous contacts, not only affects the catalytic properties of long/extralong MnPs but is also associated with their high acidic stability. Cd2+ binds at the Mn2+-oxidation site and competitively inhibits oxidation of both Mn2+ and ABTS. Moreover, mutations blocking the haem-propionate channel prevent substrate oxidation. This agrees with molecular simulations that position ABTS at an electron-transfer distance from the haem propionates of an in silico shortened-tail form, while it cannot reach this position in the extralong MnP crystal structure. Only small differences exist between the long and the extralong MnPs, which do not justify their classification as two different subfamilies, but they significantly differ from the short MnPs, with the presence/absence of the C-terminal tail extension being implicated in these differences.

Peroxygenases are very promising catalysts for oxyfunctionalization reactions. Their practical applicability, however, is hampered by their sensitivity against the oxidant (H2O2), therefore necessitating in situ generation of H2O2. Here, we report a photoelectrochemical approach to provide peroxygenases with suitable amounts of H2O2 while reducing the electrochemical overpotential needed for the reduction of molecular oxygen to H2O2. When tethered on single-walled carbon nanotubes (SWNTs) under illumination, flavins allowed for a marked anodic shift of the oxygen reduction potential in comparison to pristine SWNT and/or nonilluminated electrodes. This flavin-SWNT-based photoelectrochemical platform enabled peroxygenases-catalyzed, selective hydroxylation reactions.

A catalytic, enzyme-initiated (aza-) Achmatowicz reaction is presented. The involvement of a robust vanadium-dependent peroxidase from Curvularia inaequalis allows the simple use of H2O2 and catalytic amounts of bromide.

Nitrile reductases catalyse a two-step reduction of nitriles to amines. This requires the binding of two NADPH molecules during one catalytic cycle. For the nitrile reductase from E. coli (EcoNR) mass spectrometry studies of the catalytic mechanism were performed. EcoNR is dimeric and has no Rossman fold. It was demonstrated that during catalysis each active site binds one substrate molecule. NADPH binds independent of the substrate. The PreQ0 binding pocket of the active site is not involved in the binding of NADPH; this is in conflict with an earlier hypothesis.

Peroxygenases catalyze a broad range of (stereo)selective oxyfunctionalization reactions. However, to access their full catalytic potential, peroxygenases need a balanced provision of hydrogen peroxide to achieve high catalytic activity while minimizing oxidative inactivation. Herein, we report an enzymatic cascade process that employs methanol as a sacrificial electron donor for the reductive activation of molecular oxygen. Full oxidation of methanol is achieved, generating three equivalents of hydrogen peroxide that can be used completely for the stereoselective hydroxylation of ethylbenzene as a model reaction. Overall we propose and demonstrate an atom-efficient and easily applicable alternative to established hydrogen peroxide generation methods, which enables the efficient use of peroxygenases for oxyfunctionalization reactions.

The vanadium-​dependent chloroperoxidase from Curvularia inaequalis is an efficient biocatalyst for the in situ generation of hypohalous acids and subsequent electrophilic oxidn.​/halogenation reactions. Esp., its superb activity and stability under operational conditions make it an attractive catalyst for org. synthesis. Herein, the efficient bromination of thymol was investigated, and turnover nos. of the enzyme were found to exceed 2 000 000. The major novelty of the work is that vanadium chloroperoxidase is more useful as a brominating enzyme than vanadium bromoperoxidase in terms of operational stability, besides being far more stable than heme-​contg. peroxidases.

28 p.-5 fig.-3 tab., Pleurotus ostreatus is an important edible mushroom and a model lignin degrading organism, whose genome contains nine genes of ligninolytic peroxidases, characteristic of white-rot fungi. These genes encode six manganese peroxidase (MnP) and three versatile peroxidase (VP) isoenzymes. Using liquid chromatography coupled to tandem mass spectrometry, secretion of four of these peroxidase isoenzymes (VP1, VP2, MnP2 and MnP6) was confirmed when P. ostreatus grows in a lignocellulose medium at 25 °C (three more isoenzymes were identified by only one unique peptide). Then, the effect of environmental parameters on the expression of the above nine genes was studied by reverse transcription-quantitative PCR by changing the incubation temperature and medium pH of P. ostreatus cultures pre-grown under the above conditions (using specific primers and two reference genes for result normalization). The cultures maintained at 25 °C (without pH adjustment) provided the highest levels of peroxidase transcripts and the highest total activity on Mn2+ (a substrate of both MnP and VP) and Reactive Black 5 (a VP specific substrate). The global analysis of the expression patterns divides peroxidase genes into three main groups according to the level of expression at optimal conditions (vp1/mnp3 > vp2/vp3/mnp1/mnp2/mnp6 > mnp4/mnp5). Decreasing or increasing the incubation temperature (to 10 °C or 37 °C) and adjusting the culture pH to acidic or alkaline conditions (pH 3 and 8) generally led to downregulation of most of the peroxidase genes (and decrease of the enzymatic activity), as shown when the transcription levels were referred to those found in the cultures maintained at the initial conditions. Temperature modification produced less dramatic effects than pH modification, with most genes being downregulated during the whole 10 °C treatment, while many of them were alternatively upregulated (often 6 h after the thermal shock) and downregulated (12 h) at 37 °C. Interestingly, mnp4 and mnp5 were the only peroxidase genes upregulated under alkaline pH conditions. The differences in the transcription levels of the peroxidase genes when the culture temperature and pH parameters were changed suggest an adaptive expression according to environmental conditions. Finally, the intracellular proteome was analyzed, under the same conditions used in the secretomic analysis, and the protein product of the highly-transcribed gene mnp3 was detected. Therefore, it was concluded that the absence of MnP3 from the secretome of the P. ostreatus lignocellulose cultures was related to impaired secretion., This work was supported by the PEROXICATS (KBBE-2010-4-265397; www.peroxicats.org) and INDOX (KBBE-2013-7-613549; www.indoxproject.eu) EU projects, and by the BIO2011-26694 and AGL2011-30495 projects of the Spanish Ministry of Economy and Competitiveness (MINECO). EF-F acknowledges a Junta de Ampliación de Estudios fellowship of the CSIC, co-funded by the European Social Fund, and FJR-D acknowledges a MINECO Ramón y Cajal contract.

[Background] The genome of Pleurotus ostreatus, an important edible mushroom and a model ligninolytic organism of interest in lignocellulose biorefineries due to its ability to delignify agricultural wastes, was sequenced with the purpose of identifying and characterizing the enzymes responsible for lignin degradation., [Results] Heterologous expression of the class II peroxidase genes, followed by kinetic studies, enabled their functional classification. The resulting inventory revealed the absence of lignin peroxidases (LiPs) and the presence of three versatile peroxidases (VPs) and six manganese peroxidases (MnPs), the crystal structures of two of them (VP1 and MnP4) were solved at 1.0 to 1.1 Å showing significant structural differences. Gene expansion supports the importance of both peroxidase types in the white-rot lifestyle of this fungus. Using a lignin model dimer and synthetic lignin, we showed that VP is able to degrade lignin. Moreover, the dual Mn-mediated and Mn-independent activity of P. ostreatus MnPs justifies their inclusion in a new peroxidase subfamily. The availability of the whole POD repertoire enabled investigation, at a biochemical level, of the existence of duplicated genes. Differences between isoenzymes are not limited to their kinetic constants. Surprising differences in their activity T50 and residual activity at both acidic and alkaline pH were observed. Directed mutagenesis and spectroscopic/structural information were combined to explain the catalytic and stability properties of the most interesting isoenzymes, and their evolutionary history was analyzed in the context of over 200 basidiomycete peroxidase sequences., [Conclusions] The analysis of the P. ostreatus genome shows a lignin-degrading system where the role generally played by LiP has been assumed by VP. Moreover, it enabled the first characterization of the complete set of peroxidase isoenzymes in a basidiomycete, revealing strong differences in stability properties and providing enzymes of biotechnological interest., This work was supported by the PEROXICATS (KBBE-2010-4-265397) and INDOX (KBBE-2013-7-613549) grants of the European Union (to ATM), by grants BIO2011-26694 (to FJR-D) and BFU2011-24615 (to AR) of the Spanish Ministry of Economy and Competitiveness (MINECO), and by US DOE grant DE-AI02-07ER64480 (to KEH). 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. AG Pisabarro (Public University of Navarre, Pamplona, Spain) is acknowledged for coordinating the P. ostreatus genome project. We thank Michael D Mozuch for assistance with the lignin depolymerization studies. EF-F acknowledges a Junta de Ampliación de Estudios fellowship of the CSIC, co-funded by the European Social Fund, and FJR-D acknowledges a MINECO Ramón y Cajal contract.

9 p.-7 fig.-2 tab.+ 7 fig. supl., Enzyme-driven oxygenation reactions are in the spotlight for organic synthesis. In this regard, a hemethiolate unspecific peroxygenase (UPO) from the fungus Chaetomium globosum has recently proven to be a suitable catalyst for selective epoxidation of unsaturated fatty acids in the context of the bio-based industry, but this enzyme could not be expressed in Escherichia coli for directed mutagenesis studies. Here, a previously unknown UPO from the related Collariella virescens (synonym: Chaetomium virescens) was obtained by E. coli expression of a putative upo gene. The activity of the purified enzyme on unsaturated fatty acids with different lengths and unsaturation degrees was tested. The ability of C. virescens UPO to epoxidize these compounds increases in the order myristoleic acid (C14:1) < palmitoleic acid (C16:1) < oleic acid (C18:1) differing from that observed for the C. globosum UPO, which also forms less hydroxylated derivatives. Given the possibility to produce the C. virescens UPO in E. coli as a recombinant enzyme and its oxyfunctionalization ability, some mutated variants were obtained mimicking the active-site of C. globosum UPO and evaluated on 18-carbon unsaturated fatty acids. Results revealed that widening the heme-access channel of C. virescens UPO by substituting a phenylalanine residue (in a F88L variant) maintains the enzyme epoxidation activity, and reduces undesired hydroxylation side-reactions (from 34% to only 7% of linoleic acid products) approaching the oxyfunctionalization pattern obtained with C. globosum UPO, although maintaining the absence of diepoxides. Conversely, its partial occlusion by introducing a second phenylalanine residue (in a T158F variant) resulted in partial selective epoxidation of linoleic acid (C18:2), while the oleic acid epoxidation was prevented. The above results show how E. coli expression can speed up the availability of new UPOs, and the design of ad hoc variants of these self-sufficient monooxygenases., This work has been funded by the SusBind (“Sustainable biobinders”; H2020-BBI-JTI-2017-792063; https://susbind.eu) project of the European BBI-JU (www.bbi-europe.eu) in the frame of EU Horizon 2020.

The peroxygenase from Agrocybe aegerita (AaeUPO) has been evaluated for stereoselective oxyfunctionalization chemistry under non-aqueous reaction conditions. The stereoselective hydroxylation of ethylbenzene to (R)-1-phenylethanol was performed in neat substrate as reaction medium together with the immobilized biocatalyst and tertBuOOH as oxidant. Stability and activity issues still have to be addressed. Nevertheless, gram-scale production of enantiopure (R)-1-phenylethanol was achieved with respectable 90,000 turnovers of the biocatalyst., Financial support by the European Research Council (ERC Consolidator Grant No. 648026) is gratefully acknowledged.

Old Yellow Enzymes (OYEs) have become practical catalysts for the stereoselective trans-hydrogenation of conjugated C=C-double bonds. Despite their importance, in-depth characterization of their kinetic parameters and factors influencing their activity and stability have largely been neglected in the past years. In this study, we close this gap for one of the most prominent OYEs, i. e. the OYE from Bacillus subtilis (YqjM).

30 p.-9 fig.-2 tab.-10 fig. supl.-3 tab. supl., 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 BBIJU (www.bbi-europe.eu) project EnzOx2 (BBIPPP-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.

Peroxygenases offer an attractive means to address challenges in selective oxyfunctionalization chemistry. Despite this, their application in synthetic chemistry remains challenging due to their facile inactivation by the stoichiometric oxidant H2O2. Often atom-inefficient peroxide generation systems are required, which show little potential for large-scale implementation. Here, we show that visible-light-driven, catalytic water oxidation can be used for in situ generation of H2O2 from water, rendering the peroxygenase catalytically active. In this way, the stereoselective oxyfunctionalization of hydrocarbons can be achieved by simply using the catalytic system, water and visible light., Financial support by the European Research Council (ERC Consolidator grant no. 648026) is gratefully acknowledged. The authors thank B. Norder for XRD, W. H. Evers for TEM and F. Hagen for EPR measurements. The authors also thank S. Schmidt for the preparation of benzaldehyde lyase, M. Pesic for the preparation of YqjM and T. Knaus for the preparation of ω-transaminases. F.G.M. received funding as an ERC Starting Grant Fellow (grant agreement 638271).

Two phylogenetically divergent genes of the new family of dye-decolorizing peroxidases (DyPs) were found during comparison of the four DyP genes identified in the Pleurotus ostreatus genome with over 200 DyP genes from other basidiomycete genomes. The heterologously expressed enzymes (Pleos-DyP1 and Pleos-DyP4, following the genome nomenclature) efficiently oxidize anthraquinoid dyes (such as Reactive Blue 19), which are characteristic DyP substrates, as well as low redox-potential dyes (such as 2,2-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid)) and substituted phenols. However, only Pleos-DyP4 oxidizes the high redox-potential dye Reactive Black 5, at the same time that it displays high thermal and pH stability. Unexpectedly, both enzymes also oxidize Mn2+ to Mn3+, albeit with very different catalytic efficiencies. Pleos-DyP4 presents a Mn2+ turnover (56 s−1) nearly in the same order of the two other Mn2+-oxidizing peroxidase families identified in the P. ostreatus genome: manganese peroxidases (100 s−1 average turnover) and versatile peroxidases (145 s−1 average turnover), whose genes were also heterologously expressed. Oxidation of Mn2+ has been reported for an Amycolatopsis DyP (24 s−1) and claimed for other bacterial DyPs, albeit with lower activities, but this is the first time that Mn2+ oxidation is reported for a fungal DyP. Interestingly, Pleos-DyP4 (together with ligninolytic peroxidases) is detected in the secretome of P. ostreatus grown on different lignocellulosic substrates. It is suggested that generation of Mn3+ oxidizers plays a role in the P. ostreatus white-rot lifestyle since three different families of Mn2+-oxidizing peroxidase genes are present in its genome being expressed during lignocellulose degradation. Electronic supplementary material The online version of this article (doi:10.1007/s00253-015-6665-3) contains supplementary material, which is available to authorized users.