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Iron-sulfur (Fe-S) clusters are ancient and ubiquitous protein cofactors and play irreplaceable roles in many metabolic and regulatory processes. Fe-S clusters are built and distributed to Fe-S enzymes by dedicated protein networks. The core components of these networks are widely conserved and highly versatile. However, Fe-S proteins and enzymes are often inactive outside their native host species. We sought to systematically investigate the compatibility of Fe-S networks with non-native Fe-S enzymes. By using collections of Fe-S enzyme orthologs representative of the entire range of prokaryotic diversity, we uncovered a striking correlation between phylogenetic distance and probability of functional expression. Moreover, coexpression of a heterologous Fe-S biogenesis pathway increases the phylogenetic range of orthologs that can be supported by the foreign host. We also find that Fe-S enzymes that require specific electron carrier proteins are rarely functionally expressed unless their taxon-specific reducing partners are identified and co-expressed. We demonstrate how these principles can be applied to improve the activity of a radical S-adenosyl methionine(rSAM) enzyme from a Streptomyces antibiotic biosynthesis pathway in Escherichia coli. Our results clarify how oxygen sensitivity and incompatibilities with foreign Fe-S and electron transfer networks each impede heterologous activity. In particular, identifying compatible electron transfer proteins and heterologous Fe-S biogenesis pathways may prove essential for engineering functional Fe-S enzyme-dependent pathways.

9 p.-8 fig.-2 tab., Unspecific peroxygenases (UPOs) are fungal secreted counterparts of the cytochrome P450 monooxygenases present in most living cells. Both enzyme types share the ability to perform selective oxygenation reactions. Moreover, the Marasmius rotula UPO (MroUPO) catalyzes reactions of interest compared with the previously described UPOs, including formation of reactive epoxy fatty acids. To investigate substrate epoxidation, the most frequent positions of oleic acid at the MroUPO heme channel were predicted using binding and molecular dynamics simulations. Then, mutations in neighbor residues were designed aiming at modulating the enzyme epoxidation vs hydroxylation ratio. Both the native (wild-type recombinant) MroUPO and the mutated variants were expressed in Escherichia coli as active enzymes, and their action on oleic and other fatty acids was investigated by gas chromatography–mass spectrometry in combination with kinetic analyses. Interestingly, a small modification of the channel shape in the I153T variant increased the ratio between epoxidized oleic acid and its additionally hydroxylated derivatives. A fully opposite effect was attained with the double I153F/S156F variant that completely abolished the ability of the MroUPO to epoxidize oleic acid (while no activity was detected for the I153V variant). The rationale for these results was revealed by the substrate positioning in the above computational simulations, which predict a shorter distance between the oleic acid double bond and the oxygen atom of the peroxide-activated heme (compound I) in the I153T variant than in the native enzyme, promoting epoxidation. In contrast, the I153F/S156F double mutation fully prevents the approach of oleic acid in the bent conformation required for double-bond epoxidation, although its (sub)terminal hydroxylation was predicted and experimentally confirmed. The I153T mutation also increased the UPO selectivity on polyunsaturated fatty acid epoxidation, strongly reducing the ratio between simple epoxides and their hydroxylated derivatives, with respect to the native UPO., This work has been funded by the BBI-JU (www.bbi-europe.eu) project SusBind (on “Sustainable biobinders”; H2020-BBI-JTI-2017-792063; https://susbind.eu), together with the CTQ2016-79138-R and BIO2017-86559-R (GENOBIOREF) projects of the Spanish Ministry of Economy, Industry and Competitiveness, cofinanced by FEDER funds., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

The selective oxidation of trans-2-hexen-1-ol to the corresponding aldehyde using a recombinant aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) is reported. Especially using the two liquid phase system to overcome solubility and product inhibition issues enabled to achieve more than 2.200.000 catalytic turnovers for the production enzyme as well as molar product concentrations, pointing towards an economic feasible reaction.

1 páginas., The authors regret that, due to a re-estimation of the molar extinction coefficient of the enzyme here described, the values of several calculations presented in the paper must be amended. Such amendments concern the enzyme concentrations used in different parts of the manuscript that, due to the erroneous coefficient value initially used, were actually several times higher, and kcat values were therefore overestimated (for clarity the correct extinction coefficient is provided in the list of amendments below). The required amendments are the following:Page 719: • Right col., 4th line: 0.25 μM should be 1.4 μM • Right col., 13th line: 0.25–0.4 μM should be 1.4–2.3 μM • Right col., 31st line: 75 nM should be to 4.3 μM Page 720: • Fig. 2 caption, 4th line: 0.25–0.4 μM should be 1.4–2.3 μM Page 721: • Left col., 3rd line: 1 μM should be 6 μM • Right col., 26th line: “despite the higher turnover number of the C. virescens enzyme” should be replaced by “for the latter enzyme” • Fig. 4 caption, 4th line: 75 nM should be 4.3 μM Page 722: • Table 1: values of kcat and kcat/Km for CviUPO and F88L should read as: CviUPO kcat: 2.2 ± 0.05 (instead of 12.6 ± 0.3); CviUPO kcat/Km: 37.4 ± 3.9 (instead of 214 ± 23) F88L kcat: 1.2 ± 0.03 (instead of 7.0 ± 0.2); F88L kcat/Km: 39.2 ± 4.8 (instead of 224 ± 27) • Footnote of Table 2: 0.25–0.4 μM should be 1.4–2.3 μM Apart from the abovementioned amendments, the authors would like to add the following sentence, which was not present in the published article, providing the correct extinction coefficient of the enzyme: • Page 718, right col., 47th line (after the last sentence of UPO purification sub-section): The molar extinction coefficient of the enzyme was estimated (ε420 nm 114 mM−1 cm−1). Finally, it is important to mention that the above changes do not affect the conclusions of the paper that (as stated in the last sentence of the Abstract) basically concern: i) the possibility to enlarge the repertoire of UPOs available by E. coli expression of putative upo genes from genomes, etc. (a qualitative observation remaining valid after the changes); and ii) the effect of heme-channel mutations on the oxygenation pattern of unsaturated fatty acids by the enzyme (with the activity ratios between mutated and wild-type enzymes remaining unchanged after the correction, as shown in Table 2, etc.). The Royal Society of Chemistry apologises for these errors and any consequent inconvenience to authors and readers. © 2020 The Royal Society of Chemistry.

7 p.-1 schem.-3 fig.-1 tab., The biocatalytic preparation of trans-hex-2-enal from trans-hex-2-enol using a novel aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) is reported. As O2-dependent enzyme PeAAOx-dependent reactions are generally plagued by the poor solubility of O2 in aqueous media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by conducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency (up to 38 s-1) and turnover numbers (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme., We thank the Netherlands Organisation for Scientific Research for financial support through a VICI grant (no. 724.014.003).

11p.-5 fig.-1 tab. + 13 p.- Supporting Information, Peroxidases are considered essential agents of lignin degradation by white-rot basidiomycetes. However, low-molecular-weight oxidants likely have a primary role in lignin breakdown because many of these fungi delignify wood before its porosity has sufficiently increased for enzymes to infiltrate. It has been proposed that lignin peroxidases (LPs, EC 1.11.1.14) fulfill this role by oxidizing the secreted fungal metabolite veratryl alcohol (VA) to its aryl cation radical (VA+•), releasing it to act as a one-electron lignin oxidant within woody plant cell walls. Here, we attached the fluorescent oxidant sensor BODIPY 581/591 throughout beads with a nominal porosity of 6 kDa and assessed whether peroxidase-generated aryl cation radical systems could oxidize the beads. As positive control, we used the 1,2,4,5-tetramethoxybenzene (TMB) cation radical, generated from TMB by horseradish peroxidase. This control oxidized the beads to depths that increased with the amount of oxidant supplied, ultimately resulting in completely oxidized beads. A reaction-diffusion computer model yielded oxidation profiles that were within the 95% confidence intervals for the data. By contrast, bead oxidation caused by VA and the LPA isozyme of Phanerochaete chrysosporium was confined to a shallow shell of LP-accessible volume at the bead surface, regardless of how much oxidant was supplied. This finding contrasted with the modeling results, which showed that if the LP/VA system were to release VA+•, it would oxidize the bead interiors. We conclude that LPA releases insignificant quantities of VA+• and that a different mechanism produces small ligninolytic oxidants during white rot., This work was supported by United States Department of Energy, Office of Biological and Environmental Research Grant DE-SC0006929 (to K. E. H.,C. J. H.,and C. G. H.)

Selektive Oxyfunktionalisierungen von inerten C-H-Bindungen konnen unter milden Bedingungen durch den Einsatz von Peroxygenasen erreicht werden. Dieser Ansatz wird jedoch durch die schlechte Stabilitat der Enzyme in Anwesenheit stochiometrischer Mengen des Oxidationsmittels Wasserstoffperoxid erschwert. Hier zeigen wir, dass sich anorganische Photokatalysatoren wie goldbeladenes Titandioxid zur effizienten Bereitstellung geeigneter Mengen an H2O2 durch reduktive Aktivierung von Umgebungssauerstoff und simultaner Oxidation von Methanol eignen und gleichzeitig eine hohe Reaktivitat und Stabilitat des Enzyms gewahrleisten. Mithilfe dieses Ansatzes konnte die stereoselektive Hydroxylierung von Ethylbenzol zu (R)-1-Phenylethanol in hoher Enantioselektivitat (>98 % ee) und sehr hoher katalytischer Produktivitat (TON>71.000) erreicht werden.

[EN] There is an increasing interest in the application of peroxygenases in biocatalysis, because of their ability to catalyse the oxyfunctionalisation reaction in a stereoselective fashion and with high catalytic efficiencies, while using hydrogen peroxide or organic peroxides as oxidant. However, enzymes belonging to this class exhibit a very low stability in the presence of peroxides. With the aim of bypassing this fast and irreversible inactivation, we study the use of a gradual supply of hydrogen peroxide to maintain its concentration at stoichiometric levels. In this contribution, we report a multienzymatic cascade for in situ generation of hydrogen peroxide. In the first step, in the presence of NAD+ cofactor, formate dehydrogenase from Candida boidinii (FDH) catalysed the oxidation of formate yielding CO2. Reduced NADH was reoxidised by the reduction of the flavin mononucleotide cofactor bound to an old yellow enzyme homologue from Bacillus subtilis (YqjM), which subsequently reacts with molecular oxygen yielding hydrogen peroxide. Finally, this system was coupled to the hydroxylation of ethylbenzene reaction catalysed by an evolved peroxygenase from Agrocybe aegerita (rAaeUPO). Additionally, we studied the influence of different reaction parameters on the performance of the cascade with the aim of improving the turnover of the hydroxylation reaction., Financial support by the European Research Council (ERC Consolidator Grant No. 648026) is gratefully acknowledged.

[EN] The E-factor has become an important measure for the environmental impact of (bio)chemical reactions. However, summing up the obvious wastes generated in the laboratory neglects energy-related wastes (mostly greenhouse gases) which are generated elsewhere. To estimate these wastes, we propose to extend the E-factor by an energy-term (E-factor). At the example of a lab-scale enzyme fermentation, we demonstrate that the E-factor can constitute a multiple of the classical E-factor and therefore must not be neglected striving for a holistic estimation of the environmental impact., This workwas supported by the European Union Project H2020-BBI-PPP-2015-2-720297-ENZOX2 and F.H. gratefully acknowledges funding by European Research Council (ERC Consolidator Grant No. 648026) and the for financial support through a Netherlands Organisation for Scientific Research VICI grant (no. 724.014.003). J.M.R, B.R and A.S.B. gratefully acknowledge support from the United States National Science Foundation grant IIP-1540017.

[EN] Peroxygenases require a controlled supply of HO to operate efficiently. Here, we propose a photocatalytic system for the reductive activation of ambient O to produce HO which uses the energy provided by visible light more efficiently based on the combination of wavelength-complementary photosensitizers. This approach was coupled to an enzymatic system to make formate available as a sacrificial electron donor. The scope and current limitations of this approach are reported and discussed., We thank The Netherlands Organisation for Scientific Research for financial support through a VICI grant (No. 724.014.003).

Background 2,5-Furandicarboxylic acid is a renewable building block for the production of polyfurandicarboxylates, which are biodegradable polyesters expected to substitute their classical counterparts derived from fossil resources. It may be produced from bio-based 5-hydroxymethylfurfural or 5-methoxymethylfurfural, both obtained by the acidic dehydration of biomass-derived fructose. 5-Methoxymethylfurfural, which is produced in the presence of methanol, generates less by-products and exhibits better storage stability than 5-hydroxymethylfurfural being, therefore, the industrial substrate of choice. Results In this work, an enzymatic cascade involving three fungal oxidoreductases has been developed for the production of 2,5-furandicarboxylic acid from 5-methoxymethylfurfural. Aryl-alcohol oxidase and unspecific peroxygenase act on 5-methoxymethylfurfural and its partially oxidized derivatives yielding 2,5-furandicarboxylic acid, as well as methanol as a by-product. Methanol oxidase takes advantage of the methanol released for in situ producing H2O2 that, along with that produced by aryl-alcohol oxidase, fuels the peroxygenase reactions. In this way, the enzymatic cascade proceeds independently, with the only input of atmospheric O2, to attain a 70% conversion of initial 5-methoxymethylfurfural. The addition of some exogenous methanol to the reaction further improves the yield to attain an almost complete conversion of 5-methoxymethylfurfural into 2,5-furandicarboxylic acid. Conclusions The synergistic action of aryl-alcohol oxidase and unspecific peroxygenase in the presence of 5-methoxymethylfurfural and O2 is sufficient for the production of 2,5-furandicarboxylic acid. The addition of methanol oxidase to the enzymatic cascade increases the 2,5-furandicarboxylic acid yields by oxidizing a reaction by-product to fuel the peroxygenase reactions., This work has been funded by the H2020 BBI-JU (http://www.bbi-europe.eu) project EnzOx2 (H2020-BBI-PPP-2015-2-720297) together with the INDOX EU project (FP7-KBBE-2013-7-613549) and the NOESIS (BIO2014-56388-R) and GENOBIOREF (BIO2017-86559-R) projects of the Spanish Ministry of Economy, Industry and Competitiveness, co-fnanced by FEDER funds., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

Oxidation chemistry using enzymes is approaching maturity and practical applicability in organic synthesis. Oxidoreductases (enzymes catalysing redox reactions) enable chemists to perform highly selective and efficient transformations ranging from simple alcohol oxidations to stereoselective halogenations of non‐activated C−H bonds. For many of these reactions, no “classical” chemical counterpart is known. Hence oxidoreductases open up shorter synthesis routes based on a more direct access to the target products. The generally very mild reaction conditions may also reduce the environmental impact of biocatalytic reactions compared to classical counterparts. In this Review, we critically summarise the most important recent developments in the field of biocatalytic oxidation chemistry and identify the most pressing bottlenecks as well as promising solutions.

A photoenzymatic NADH regeneration system was established. The combination of deazariboflavin as a photocatalyst with putidaredoxin reductase enabled the selective reduction of NAD+ into the enzyme-active 1,4-NADH to promote an alcohol dehydrogenase catalysed stereospecific reduction reaction. The catalytic turnover of all the reaction components was demonstrated. Factors influencing the efficiency of the overall system were identified.

Peroxygenasen katalysieren eine Vielzahl stereoselektiver Oxyfunktionalisierungen unter Verwendung von Wasserstoffperoxid als einzigem Oxidationsmittel. Allerdings werden Peroxygenasen auch durch H2O2 irreversibel inaktiviert. Fur robuste Peroxygenase-Reaktionen ist also eine effiziente In-situ-Bereitstellung von H2O2 unabdingbar. Hier beschreiben wir eine neuartige Enzymkaskade zur reduktiven Aktivierung von Luftsauerstoff unter Verwendung von Methanol als stochiometrischem Reduktionsmittel. Am Beispiel der stereoselektiven Hydroxylierung von Ethylbenzol zu (R)-1-Phenylethanol zeigen wir die effiziente Nutzung aller in Methanol gespeicherten Reduktionsaquivalente zur reduktiven Aktivierung von O2.

The biocatalytic preparation of trans-hex-2-enal from trans-hex-2-enol using a novel aryl alcohol oxidase from Pleurotus eryngii (PeAAOx) is reported. As O2-dependent enzyme PeAAOx-dependent reactions are generally plagued by the poor solubility of O2 in aqueous media and mass transfer limitations resulting in poor reaction rates. These limitations were efficiently overcome by conducting the reaction in a flow-reactor setup reaching unpreceded catalytic activities for the enzyme in terms of turnover frequency (up to 38 s−1) and turnover numbers (more than 300000) pointing towards preparative usefulness of the proposed reaction scheme.

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.

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.

18 p.-8 fig., [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., We acknowledge support by the CSIC Open Access Publication Initiative through its Unit of Information Resources for Research (URICI).

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.