@misc{KinnePorajKobielskaUllrichetal., author = {Kinne, Matthias and Poraj-Kobielska, Marzena and Ullrich, Ren{\´e} and Nousiainen, Paula and Sipil{\"a}, Jussi and Scheibner, Katrin and Hammel, Kenneth E. and Hofrichter, Martin}, title = {Oxidative cleavage of non-phenolic b-O-4 lignin model dimers by an extracellular aromatic peroxygenase}, series = {Holzforschung}, volume = {65}, journal = {Holzforschung}, number = {5}, issn = {1437-434X}, doi = {10.1515/HF.2011.057}, pages = {673 -- 679}, abstract = {The extracellular aromatic peroxygenase of the agaric fungus Agrocybe aegerita catalyzed the H2O2-dependent cleavage of non-phenolic arylglycerol-b-aryl ethers (b-O-4 ethers). For instance 1-(3,4-dimethoxyphenyl)-2-(2-methoxy-phenoxy)pro- pane-1,3-diol, a recalcitrant dimeric lignin model compound that represents the major non-phenolic substructure in lignin, was selectively O-demethylated at the para-methoxy group to give formaldehyde and 1-(4-hydroxy-3-methoxyphenyl)- 2-(2-methoxyphenoxy)propane-1,3-diol. The phenol moiety of the latter compound was then enzymatically oxidized into phenoxy radicals and a quinoid cation, which initiated the autocatalytic cleavage of the dimer and the formation of monomers such as 2-methoxy-1,4-benzoquinone and phenoxyl-substituted propionic acid. The introduction of 18O from H218O2 and H218O at different positions into the products provided information about the routes of ether cleavage. Studies with a 14C-labeled lignin model dimer showed that more than 70\% of the intermediates formed were further coupled to form polymers with molecular masses above 10 kDa. The results indicate that fungal aromatic peroxyge- nases may be involved in the bioconversion of methoxylated plant ingredients originating from lignin or other sources.}, language = {en} } @misc{YarmanPengWuetal., author = {Yarman, Aysu and Peng, Lei and Wu, Yunhua and Bandodkar, Amay and Gajovic-Eichelmann, Nenad and Wollenberger, Ulla and Hofrichter, Martin and Ullrich, Ren{\´e} and Scheibner, Katrin and Scheller, Frieder W.}, title = {Can peroxygenase and microperoxidase substitute cytochrome P450 in biosensors}, series = {Bioanalytical Reviews}, volume = {3}, journal = {Bioanalytical Reviews}, number = {2-4}, issn = {1867-2094}, doi = {10.1007/s12566-011-0023-4}, pages = {67 -- 94}, abstract = {Aromatic peroxygenase (APO) from the basidiomycetous mushroom Agrocybe aegerita (AaeAPO) and microperoxidases (MPs) obtained from cytochrome c exhibit a broad substrate spectrum including hydroxylation of selected aromatic substrates, demethylation and epoxidation by means of hydrogen peroxide. It overlaps with that of cytochrome P450 (P450), making MPs and APOs to alternate recognition elements in biosensors for the detection of typical P450 substrates. Here, we discuss recently developed approaches using microperoxidases and peroxygenases in view of their potential to supplement P450 enzymes as recognition elements in biosensors for aromatic compounds. Starting as early as the 1970s, the direct electron transfer between electrodes and the heme group of heme peptides called microperoxidases has been used as a model of oxidoreductases. These MP-modified electrodes are used as hydrogen peroxide detectors based on the catalytic current generated by electrically contacted microperoxidase molecules. A similar catalytic reaction has been obtained for the electrode-immobilised heme protein AaeAPO. However, up to now, no MP-based sensors for substrates have been described. In this review, we present biosensors which indicate 4-nitrophenol, aniline, naphthalene and p-aminophenol based on the peroxide-dependent substrate conversion by electrode-immobilised MP and AaeAPO. In these enzyme electrodes, the signal is generated by the conversion of all substrates, thus representing in complex media an overall parameter. The performance of these sensors and their further development are discussed in comparison with P450-based electrodes.}, language = {en} } @misc{PorajKobielskaKinneUllrichetal., author = {Poraj-Kobielska, Marzena and Kinne, Matthias and Ullrich, Ren{\´e} and Scheibner, Katrin and Kayser, Gernot and Hammel, Kenneth E. and Hofrichter, Martin}, title = {Preparation of human drug metabolites using fungal peroxygenases}, series = {Biochemical Pharmacology}, volume = {82}, journal = {Biochemical Pharmacology}, number = {7}, issn = {1873-2968}, doi = {10.1016/j.bcp.2011.06.020}, pages = {789 -- 796}, abstract = {The synthesis of hydroxylated and O- or N-dealkylated human drug metabolites (HDMs) via selective monooxygenation remains a challenging task for synthetic organic chemists. Here we report that aromatic peroxygenases (APOs; EC 1.11.2.1) secreted by the agaric fungi Agrocybe aegerita and Coprinellus radians catalyzed the H₂O₂-dependent selective monooxygenation of diverse drugs, including acetanilide, dextrorphan, ibuprofen, naproxen, phenacetin, sildenafil and tolbutamide. Reactions included the hydroxylation of aromatic rings and aliphatic side chains, as well as O- and N-dealkylations and exhibited different regioselectivities depending on the particular APO used. At best, desired HDMs were obtained in yields greater than 80\% and with isomeric purities up to 99\%. Oxidations of tolbutamide, acetanilide and carbamazepine in the presence of H₂¹⁸O₂ resulted in almost complete incorporation of ¹⁸O into the corresponding products, thus establishing that these reactions are peroxygenations. The deethylation of phenacetin-d₁ showed an observed intramolecular deuterium isotope effect [(k(H)/k(D))(obs)] of 3.1±0.2, which is consistent with the existence of a cytochrome P450-like intermediate in the reaction cycle of APOs. Our results indicate that fungal peroxygenases may be useful biocatalytic tools to prepare pharmacologically relevant drug metabolites.}, language = {en} } @misc{PengWollenbergerKinneetal., author = {Peng, Lei and Wollenberger, Ulla and Kinne, Matthias and Hofrichter, Martin and Ullrich, Ren{\´e} and Scheibner, Katrin and Fischer, Anna and Scheller, Frieder W.}, title = {Peroxygenase based sensor for aromatic compounds}, series = {Biosensors and Bioelectronics}, volume = {26}, journal = {Biosensors and Bioelectronics}, number = {4}, issn = {1873-4235}, doi = {10.1016/j.bios.2010.07.075}, url = {http://nbn-resolving.de/http://www.sciencedirect.com/science/article/pii/S0956566310004501}, pages = {1432 -- 1436}, abstract = {We report on the redox behaviour of the peroxygenase from Agrocybe aegerita (AaeAPO) which has been electrostatically immobilized in a matrix of chitosan-embedded gold nanoparticles on the surface of a glassy carbon electrode. AaeAPO contains a covalently bound heme-thiolate as the redox active group that exchanges directly electrons with the electrode via the gold nanoparticles. The formal potential E ° ′ of AaeAPO in the gold nanoparticles-chitosan film was estimated to be -(286 ± 9) mV at pH 7.0. The heterogeneous electron transfer rate constant (ks) increases from 3.7 in the scan rate range from 0.2 to 3.0 V s-1 and level off at 63.7 s-1. Furthermore, the peroxide-dependent hydroxylation of aromatic compounds was applied to develop a sensor for naphthalene and nitrophenol. The amperometric measurements of naphthalene are based on the indication of H2O2 consumption. For the chitosan-embedded gold nanoparticle system, the linear range extends from 4 to 40 μM naphthalene with a detection limit of 4.0 μM (S/N = 3) and repeatability of 5.7\% for 40 μM naphthalene.}, language = {en} } @misc{PengWollenbergerHofrichteretal., author = {Peng, Lei and Wollenberger, Ulla and Hofrichter, Martin and Ullrich, Ren{\´e} and Scheibner, Katrin and Scheller, Frieder W.}, title = {Bioelectrocatalytic properties of Agrocybe aegerita peroxygenase}, series = {Electrochimica Acta}, volume = {55}, journal = {Electrochimica Acta}, number = {27}, issn = {0013-4686}, doi = {10.1016/j.electacta.2009.12.065}, pages = {7809 -- 7813}, abstract = {A biosensor for detecting the aromatic substance 4-nitrophenol based on Agrocybe aegerita peroxygenase (AaP) immobilized with chitosan-stabilized gold nanoparticles is presented here. This biosensor measures the enzymatic product of 4-nitrophenol peroxygenation, 4-nitrocatechol, which is electrochemically detected in the presence of hydrogen peroxide. Cyclic voltammetry and amperometry were used to characterize the proposed biosensor. The linear range of the AaP biosensor for the detection of 4-nitrophenol was between 10 and 30 μM with a detection limit of 0.2 μM (based on the S/N = 3). The catalytic property of AaP to oxidize 4-nitrophenol was compared with two other heme proteins, a camphor-hydroxylating cytochrome P450 monooxygenase (P450cam, CYP101) and horseradish peroxidase (HRP). The results revealed that only AaP is capable of catalyzing the hydroxylation of 4-nitrophenol into 4-nitrocatechol. Consequently, AaP could be a particularly potent biocatalyst that may fill the gap between cytochrome P450s and common heme peroxidases.}, language = {en} } @misc{PecynaUllrichBittneretal., author = {Pecyna, Marek J. and Ullrich, Ren{\´e} and Bittner, Britta and Clemens, Andr{\´e} and Scheibner, Katrin and Schubert, Roland and Hofrichter, Martin}, title = {Molecular characterization of aromatic peroxygenase from Agrocybe aegerita}, series = {Applied Microbiology and Biotechnology}, volume = {84}, journal = {Applied Microbiology and Biotechnology}, number = {5}, issn = {1432-0614}, doi = {10.1007/s00253-009-2000-1}, pages = {885 -- 897}, abstract = {Recently, a novel group of fungal peroxidases, known as the aromatic peroxygenases (APO), has been discovered. Members of these extracellular biocatalysts produced by agaric basidiomycetes such as Agrocybe aegerita or Coprinellus radians catalyze reactions—for example, the peroxygenation of naphthalene, toluene, dibenzothiophene, or pyridine—which are actually attributed to cytochrome P450 monooxygenases. Here, for the first time, genetic information is presented on this new group of peroxide-consuming enzymes. The gene of A. aegerita peroxygenase (apo1) was identified on the level of messenger RNA and genomic DNA. The gene sequence was affirmed by peptide sequences obtained through an Edman degradation and de novo peptide sequencing of the purified enzyme. Quantitative real-time reverse transcriptase polymerase chain reaction demonstrated that the course of enzyme activity correlated well with that of mRNA signals for apo1 in A. aegerita. The full-length sequences of A. aegerita peroxygenase as well as a partial sequence of C. radians peroxygenase confirmed the enzymes' affiliation to the heme-thiolate proteins. The sequences revealed no homology to classic peroxidases, cytochrome P450 enzymes, and only little homology (<30\%) to fungal chloroperoxidase produced by the ascomycete Caldariomyces fumago (and this only in the N-terminal part of the protein comprising the heme-binding region and part of the distal heme pocket). This fact reinforces the novelty of APO proteins. On the other hand, homology retrievals in genetic databases resulted in the identification of various APO homologous genes and transcripts, particularly among the agaric fungi, indicating APO's widespread occurrence in the fungal kingdom.}, language = {en} } @misc{KinnePorajKobielskaArandaetal., author = {Kinne, Matthias and Poraj-Kobielska, Marzena and Aranda, Elisabet and Ullrich, Ren{\´e} and Hammel, Kenneth E. and Scheibner, Katrin and Hofrichter, Martin}, title = {Regioselective preparation of 5-hydroxypropranolol and 4′-hydroxydiclofenac with a fungal peroxygenase}, series = {Bioorganic \& Medicinal Chemistry Letters}, volume = {19}, journal = {Bioorganic \& Medicinal Chemistry Letters}, number = {11}, issn = {1464-3405}, doi = {10.1016/j.bmcl.2009.04.015}, pages = {3085 -- 3087}, abstract = {An extracellular peroxygenase of Agrocybe aegerita catalyzed the H2O2-dependent hydroxylation of the multi-function beta-adrenergic blocker propranolol (1-naphthalen-1-yloxy-3-(propan-2-ylamino)propan-2-ol) and the non-steroidal anti-inflammatory drug diclofenac (2-[2-[(2,6-dichlorophenyl)amino]phenyl]acetic acid) to give the human drug metabolites 5-hydroxypropranolol (5-OHP) and 4′-hydroxydiclofenac (4′-OHD). The reactions proceeded regioselectively with high isomeric purity and gave the desired 5-OHP and 4′-OHD in yields up to 20\% and 65\%, respectively. 18O-labeling experiments showed that the phenolic hydroxyl groups in 5-OHP and 4′-OHD originated from H2O2, which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for versatile, cost-effective, and scalable syntheses of drug metabolites.}, language = {en} } @misc{KlugeUllrichDolgeetal., author = {Kluge, Martin and Ullrich, Ren{\´e} and Dolge, Christoph and Scheibner, Katrin and Hofrichter, Martin}, title = {Hydroxylation of naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via oxygen transfer from H2O2 and intermediary epoxidation}, series = {Applied Microbiology and Biotechnology}, volume = {81}, journal = {Applied Microbiology and Biotechnology}, number = {6}, issn = {0175-7598}, doi = {10.1007/s00253-008-1704-y}, pages = {1071 -- 1076}, abstract = {Agrocybe aegerita peroxidase/peroxygenase (AaP) is an extracellular fungal biocatalyst that selectively hydroxylates the aromatic ring of naphthalene. Under alkaline conditions, the reaction proceeds via the formation of an intermediary product with a molecular mass of 144 and a characteristic UV absorption spectrum (A max 210, 267, and 303 nm). The compound was semistable at pH 9 but spontaneously hydrolyzed under acidic conditions (pH <7) into 1-naphthol as major product and traces of 2-naphthol. Based on these findings and literature data, we propose naphthalene 1,2-oxide as the primary product of AaP-catalyzed oxygenation of naphthalene. Using 18O-labeled hydrogen peroxide, the origin of the oxygen atom transferred to naphthalene was proved to be the peroxide that acts both as oxidant (primary electron acceptor) and oxygen source.}, language = {en} } @misc{KinneUllrichHammeletal., author = {Kinne, Matthias and Ullrich, Ren{\´e} and Hammel, Kenneth E. and Scheibner, Katrin and Hofrichter, Martin}, title = {Regioselective preparation of (R)-2-(4-Hydroxyphenoxy)propionic acid with a fungal peroxygenase}, series = {Tetrahedron Letters}, volume = {49}, journal = {Tetrahedron Letters}, number = {41}, issn = {1873-3581}, doi = {10.1016/j.tetlet.2008.07.152}, pages = {5950 -- 5953}, abstract = {The extracellular heme-thiolate peroxygenase of Agrocybe aegerita catalyzed the H2O2-dependent hydroxylation of 2-phenoxypropionic acid (POPA) to give the herbicide precursor 2-(4-hydroxyphenoxy)propionic acid (HPOPA). The reaction proceeded regioselectively with an isomeric purity near 98\%, and yielded the desired R-isomer of HPOPA with an enantiomeric excess of 60\%. 18O-labeling experiments showed that the phenolic hydroxyl in HPOPA originated from H2O2, which establishes that the reaction is mechanistically a peroxygenation. Our results raise the possibility that fungal peroxygenases may be useful for a variety of organic oxidations.}, language = {en} } @misc{GroebeUllrichPecynaetal., author = {Gr{\"o}be, Glenn and Ullrich, Ren{\´e} and Pecyna, Marek J. and Kapturska, Danuta and Friedrich, Stephanie and Hofrichter, Martin and Scheibner, Katrin}, title = {High-yield production of aromatic peroxygenase by the agaric fungus Marasmius rotula}, series = {AMB Express}, journal = {AMB Express}, issn = {2191-0855}, abstract = {An extracellular peroxygenase from Marasmius rotula was produced in liquid culture, chromatographically purified and partially characterized. This is the third aromatic peroxygenase (APO) that has been characterized in detail and the first one that can be produced in high yields. The highest enzyme levels of about 41,000 U l-1 (corresponding to appr. 445 mg l-1 APO protein) exceeded the hitherto reported levels more than 40-fold and were detected in carbon- and nitrogen-rich complex media. The enzyme was purified by FPLC to apparent homogeneity (SDS-PAGE) with a molecular mass of 32 kDa (27 kDa after deglycosylation) and isoelectric points between 4.97 and 5.27. The UV-visible spectrum of the native enzyme showed a characteristic maximum (Soret band) at 418 nm that shifted after reduction with sodium dithionite and flushing with carbon monoxide to 443 nm. The pH optimum of the M. rotula enzyme was found to vary between pH 5 and 6 for most reactions studied. The apparent Km-values for 2,6-dimethoxyphenol, benzyl alcohol, veratryl alcohol, naphthalene and H2O2 were 0.133, 0.118, 0.279, 0.791 and 3.14 mM, respectively. M. rotula APO was found to be highly stable in a pH range from 5 to 10 as well as in the presence of organic solvents (50\% vol/vol) such as methanol, acetonitrile and N,N-dimethylformamide. Unlike other APOs, the peroxygenase of M. rotula showed neither brominating nor chlorinating activities.}, language = {en} } @misc{FriedrichGroebeKlugeetal., author = {Friedrich, Stephanie and Gr{\"o}be, Glenn and Kluge, Martin and Brinkmann, Tobias and Hofrichter, Martin and Scheibner, Katrin}, title = {Optimization of a biocatalytic process to gain (R)-1-phenylethanol by applying the software tool Sabento for ecological assessment during the early stages of development}, series = {Journal of Molecular Catalysis : B, Enzymatic}, journal = {Journal of Molecular Catalysis : B, Enzymatic}, number = {103}, issn = {1381-1177}, doi = {10.1016/j.molcatb.2013.10.002}, pages = {36 -- 40}, abstract = {Ecological assessment using the software tool Sabento was conducted to compare different processes to gain the fine chemical (R)-1-phenylethanol from ethylbenzene. The software was applied during the biocatalytic process development using the unspecific peroxygenase (EC .11.2.1) of the fungus Agrocybe aegerita. The process could be systematically improved with respect to the ecological performance during process development. Compared to a modern chemical process and a further biotechnological process, it now reaches the best environmental key indicator. The software tool Sabento proved to be well suited to work out the most important factors determining the ecological burdens in the early stages of process development.}, language = {en} } @misc{YarmanGroebeNeumannetal., author = {Yarman, Aysu and Gr{\"o}be, Glenn and Neumann, Bettina and Kinne, Mathias and Gajovic-Eichelmann, Nenad and Wollenberger, Ulla and Hofrichter, Martin and Ullrich, Ren{\´e} and Scheibner, Katrin and Scheller, Frieder W.}, title = {The aromatic peroxygenase from Marasmius rutola—a new enzyme for biosensor applications}, series = {Analytical and Bioanalytical Chemistry}, volume = {402}, journal = {Analytical and Bioanalytical Chemistry}, number = {1}, issn = {1618-2650}, doi = {10.1007/s00216-011-5497-y}, pages = {405 -- 412}, abstract = {The aromatic peroxygenase (APO; EC 1.11.2.1) from the agraric basidomycete Marasmius rotula (MroAPO) immobilized at the chitosan-capped gold-nanoparticle-modified glassy carbon electrode displayed a pair of redox peaks with a midpoint potential of -278.5 mV vs. AgCl/AgCl (1 M KCl) for the Fe2+/Fe3+ redox couple of the heme-thiolate-containing protein. MroAPO oxidizes aromatic substrates such as aniline, p-aminophenol, hydroquinone, resorcinol, catechol, and paracetamol by means of hydrogen peroxide. The substrate spectrum overlaps with those of cytochrome P450s and plant peroxidases which are relevant in environmental analysis and drug monitoring. In M. rotula peroxygenase-based enzyme electrodes, the signal is generated by the reduction of electrode-active reaction products (e.g., p-benzoquinone and p-quinoneimine) with electro-enzymatic recycling of the analyte. In these enzyme electrodes, the signal reflects the conversion of all substrates thus representing an overall parameter in complex media. The performance of these sensors and their further development are discussed.}, language = {en} } @misc{PorajKobielskaAtzrodtHollaetal., author = {Poraj-Kobielska, Marzena and Atzrodt, Jens and Holla, Wolfgang and Sandvoss, Martin and Gr{\"o}be, Glenn and Scheibner, Katrin and Hofrichter, Martin}, title = {Preparation of labeled human drug metabolites and drug-drug interaction-probes with fungal peroxygenases}, series = {Journal of Labelled Compounds and Radiopharmaceuticals}, volume = {56}, journal = {Journal of Labelled Compounds and Radiopharmaceuticals}, number = {9-10}, issn = {1099-1344}, doi = {10.1002/jlcr.3103}, pages = {513 -- 519}, abstract = {Enzymatic conversion of a drug can be an efficient alternative for the preparation of a complex metabolite compared with a multi-step chemical synthesis approach. Limitations exist for chemical methods for direct oxygen incorporation into organic molecules often suffering from low yields and unspecific oxidation and also for alternative whole-cell biotransformation processes, which require specific fermentation know-how. Stable oxygen-transferring biocatalysts such as unspecific peroxygenases (UPOs) could be an alternative for the synthesis of human drug metabolites and related stable isotope-labeled analogues. This work shows that UPOs can be used in combination with hydrogen/deuterium exchange for an efficient one-step process for the preparation of 4'-OH-diclofenac-d6. The scope of the reaction was investigated by screening of different peroxygenase subtypes for the transformation of selected deuterium-labeled substrates such as phenacetin-d3 or lidocaine-d3. Experiments with diclofenac-d7 revealed that the deuterium-labeling does not affect the kinetic parameters. By using the latter substrate and H2 (18) O2 as cosubstrate, it was possible to prepare a doubly isotope-labeled metabolite (4'-(18) OH-diclofenac-d6). UPOs offer certain practical advantages compared with P450 enzyme systems in terms of stability and ease of handling. Given these advantages, future work will expand the existing 'monooxygenation toolbox' of different fungal peroxygenases that mimic P450 in vitro reactions.}, language = {en} } @misc{PeterKarichUllrichetal., author = {Peter, Sebastian and Karich, Alexander and Ullrich, Ren{\´e} and Gr{\"o}be, Glenn and Scheibner, Katrin and Hofrichter, Martin}, title = {Enzymatic one-pot conversion of cyclohexane into cyclohexanone: Comparison of four fungal peroxygenases}, series = {Journal of Molecular Catalysis : B, Enzymatic}, journal = {Journal of Molecular Catalysis : B, Enzymatic}, number = {103}, doi = {10.1016/j.molcatb.2013.09.016}, pages = {47 -- 51}, abstract = {Unspecific peroxygenases (UPO; EC 1.11.2.1) represent a group of secreted heme-thiolate proteins that are capable of catalyzing the mono-oxygenation of diverse organic compounds, using only H2O2 as a co-substrate. Here we show that the four peroxygenases AaeUPO, MroUPO, rCciUPO and rNOVO catalyze the stepwise hydroxylation of cyclohexane to cyclohexanol and cyclohexanone. The catalytic efficiencies (kcat/Km) for the initial hydroxylation were in the same order of magnitude for all four peroxygenases (∼104 M-1 s-1), whereas they differed in the second step. The conversion of cyclohexanol by AaeUPO and rCciUPO was 1-2 orders of magnitude less efficient (∼102 M-1 s-1) than by MroUPO and rNOVO (∼104 M-1 s-1). The highest conversion rate in terms of H2O2 utilization was accomplished by MroUPO under repeated addition of the peroxide (87\% in relation to the total products formed). Using the latter UPO, we successfully established a micro-mixing reaction device (SIMM-V2) for the oxidation of cyclohexane. As cyclohexanone is a chemical of high relevance, for example, as starting material for polymer syntheses or as organic solvent, new enzymatic production pathways for this compound are of interest to complement existing chemical and biotechnological approaches. Stable and versatile peroxygenases, as those presented here, may form a promising biocatalytic platform for the development of such enzyme-based processes.}, language = {en} } @misc{KlugeUllrichScheibneretal., author = {Kluge, Martin and Ullrich, Ren{\´e} and Scheibner, Katrin and Hofrichter, Martin}, title = {Formation of naphthalene hydrates in the enzymatic conversion of 1,2-dihydronaphthalene by two fungal peroxygenases and subsequent naphthalene formation}, series = {Journal of Molecular Catalysis : B, Enzymatic}, journal = {Journal of Molecular Catalysis : B, Enzymatic}, number = {103}, issn = {1381-1177}, doi = {10.1016/j.molcatb.2013.08.017}, pages = {56 -- 60}, abstract = {The formation of naphthalene hydrates (i.e. 1- and 2-hydroxy-1,2-dihydronaphthalene) displays a new activity (besides epoxidation) in the enzymatic transformation of 1,2-dihydronaphthalene by two fungal unspecific peroxygenases (UPOs) accounting for 16-19\% of the overall turnover. These arene hydrates decayed into naphthalene that in turn was converted by UPOs into naphthols. The oxygen transferred during hydroxylation was shown to derive from hydrogen peroxide proving a true peroxygenation reaction.}, language = {en} } @misc{KlugeUllrichScheibneretal., author = {Kluge, Martin and Ullrich, Ren{\´e} and Scheibner, Katrin and Hofrichter, Martin}, title = {Stereoselective benzylic hydroxylation of alkylbenzenes and epoxidation of styrene derivatives catalyzed by the peroxygenase of Agrocybe aegerita}, series = {Green Chemistry}, journal = {Green Chemistry}, number = {2}, issn = {1463-9270}, pages = {440 -- 446}, abstract = {Here we report on the stereoselective benzylic hydroxylation and C1-C2 epoxidation of alkylbenzenes and styrene derivatives, respectively, by a heme-thiolate peroxygenase (EC 1.11.2.1) from the fungus Agrocybe aegerita. Benzylic hydroxylation led exclusively to the (R)-1-phenylalkanols. For (R)-1-phenylethanol, (R)-1-phenylpropanol and (R)-1-tetralol, the ee reached >99\%. For longer chain lengths, the enantiomeric excesses (ee) and total turnover numbers (TTN) decreased while the number of by-products, e.g. 1-phenylketones, increased. Epoxidation of straight chain and cyclic styrene derivatives gave a heterogeneous picture and resulted in moderate to excellent ee values and TTN: e.g., in the case of (1R,2S)-cis-[small beta]-methylstyrene oxide formation, an ee >99\% and a TTN of 110 000 was achieved. Hydroxylation and epoxidation were true peroxygenations, which was demonstrated by the incorporation of 18O from H218O2 into the products. The use of fed-batch devices and varying feeding strategies for the substrate and co-substrate turned out to be a suitable approach to optimize peroxygenase catalysis.}, language = {en} } @misc{PorajKobielskaKinneUllrichetal., author = {Poraj-Kobielska, Marzena and Kinne, Matthias and Ullrich, Ren{\´e} and Scheibner, Katrin and Hofrichter, Martin}, title = {A spectrophotometric assay for the detection of fungal peroxygenases}, series = {Analytical Biochemistry}, volume = {421}, journal = {Analytical Biochemistry}, number = {1}, doi = {10.1016/j.ab.2011.10.009}, pages = {327 -- 329}, abstract = {Rapid and simple spectrophotometric methods are required for the unambiguous detection of recently discovered fungal peroxygenases in vivo and in vitro. This paper describes a peroxygenase-specific assay using 5-nitro-1,3-benzodioxole as substrate. The product, 4-nitrocatechol, produces a yellow color at pH 7, which can be followed over time at 425 nm (ε425 = 9,700 M-1 cm-1), and a red color when adjusted to pH >12, which can be measured in form of an end-point determination at 514 nm (ε514 = 11,400 M-1 cm-1). The assay is suitable for detecting peroxygenase activities in complex growth media and environmental samples as well as for high-throughput screenings.}, language = {en} } @misc{KlugeUllrichScheibneretal., author = {Kluge, Martin and Ullrich, Ren{\´e} and Scheibner, Katrin and Hofrichter, Martin}, title = {Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase-peroxygenase}, series = {Applied Microbiology and Biotechnology}, volume = {75}, journal = {Applied Microbiology and Biotechnology}, number = {6}, issn = {1432-0614}, doi = {10.1007/s00253-007-0942-8}, pages = {1473 -- 1478}, abstract = {Agrocybe aegerita peroxidase (AaP) is a versatile heme-thiolate protein that can act as a peroxygenase and catalyzes, among other reactions, the hydroxylation of aromatic rings. This paper reports a rapid and selective spectrophotometric method for directly detecting aromatic hydroxylation by AaP. The weakly activated aromatic compound naphthalene served as the substrate that was regioselectively converted into 1-naphthol in the presence of the co-substrate hydrogen peroxide. Formation of 1-naphthol was followed at 303 nm (ɛ 303 = 2,010 M-1 cm-1), and the apparent Michaelis-Menten (K m) and catalytic (k cat) constants for the reaction were estimated to be 320 μM and 166 s-1, respectively. This method will be useful in screening of fungi and other microorganisms for extracellular peroxygenase activities and in comparing and assessing different catalytic activities of haloperoxidase-peroxygenases.}, language = {en} } @misc{UllrichNueskeScheibneretal., author = {Ullrich, Ren{\´e} and N{\"u}ske, J{\"o}rg and Scheibner, Katrin and Spantzel, J{\"o}rg and Hofrichter, Martin}, title = {Novel Haloperoxidase from the Agaric Basidiomycete Agrocybe aegerita Oxidizes Aryl Alcohols and Aldehydes}, series = {Applied and Environmental Microbiology}, volume = {70}, journal = {Applied and Environmental Microbiology}, number = {8}, issn = {1098-5336}, doi = {10.1128/AEM.70.8.4575-4581.2004}, pages = {4575 -- 4581}, abstract = {Agrocybe aegerita, a bark mulch- and wood-colonizing basidiomycete, was found to produce a peroxidase (AaP) that oxidizes aryl alcohols, such as veratryl and benzyl alcohols, into the corresponding aldehydes and then into benzoic acids. The enzyme also catalyzed the oxidation of typical peroxidase substrates, such as 2,6-dimethoxyphenol (DMP) or 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonate) (ABTS). A. aegerita peroxidase production depended on the concentration of organic nitrogen in the medium, and highest enzyme levels were detected in the presence of soybean meal. Two fractions of the enzyme, AaP I and AaP II, which had identical molecular masses (46 kDa) and isoelectric points of 4.6 to 5.4 and 4.9 to 5.6, respectively (corresponding to six different isoforms), were identified after several steps of purification, including anion- and cation-exchange chromatography. The optimum pH for the oxidation of aryl alcohols was found to be around 7, and the enzyme required relatively high concentrations of H2O2 (2 mM) for optimum activity. The apparent Km values for ABTS, DMP, benzyl alcohol, veratryl alcohol, and H2O2 were 37, 298, 1,001, 2,367 and 1,313 μM, respectively. The N-terminal amino acid sequences of the main AaP II spots blotted after two-dimensional gel electrophoresis were almost identical and exhibited almost no homology to the sequences of other peroxidases from basidiomycetes, but they shared the first three amino acids, as well as two additional amino acids, with the heme chloroperoxidase (CPO) from the ascomycete Caldariomyces fumago. This finding is consistent with the fact that AaP halogenates monochlorodimedone, the specific substrate of CPO. The existence of haloperoxidases in basidiomycetous fungi may be of general significance for the natural formation of chlorinated organic compounds in forest soils.}, language = {en} } @misc{MartinezRuizDuenasGutierrezetal., author = {Mart{\´i}nez, Angel T. and Ruiz-Due{\~n}as, Francisco J. and Guti{\´e}rrez, Ana and R{\´i}o, Jos{\´e} C. del and Alcalde, Miguel and Liers, Christiane and Ullrich, Ren{\´e} and Hofrichter, Martin and Scheibner, Katrin and Kalum, Lisbeth and Vind, Jesper and Lund, Henrik}, title = {Search, engineering, and applications of new oxidative biocatalysts}, series = {Biofuels, Bioproducts and Biorefining}, volume = {8}, journal = {Biofuels, Bioproducts and Biorefining}, number = {6}, issn = {1932-1031}, doi = {10.1002/bbb.1498}, pages = {819 -- 835}, abstract = {Most industrial enzymes are hydrolases, such as glycosidases and esterases. However, oxidoreductases have an unexploited potential for substituting harsh (and scarcely selective) chemical processes. A group of basidiomycetes are the only organisms degrading the aromatic lignin polymer, enabling the subsequent use of plant polysaccharides. Therefore, these fungi and their ligninolytic peroxidases are the biocatalysts of choice for industrial delignification and oxidative biotransformations of aromatic and other organic compounds. The latter also include oxygenation reactions, which are catalyzed with high regio/stereo selectivity by fungal peroxygenases. In search for novel and more robust peroxidases/peroxygenases, basidiomycetes from unexplored habitats were screened, and hundreds of genes identified in basidiomycete genomes (in collaboration with the DOE JGI). The most interesting genes were heterologously expressed, and the corresponding enzymes structurally-functionally characterized. The information obtained enabled us to improve the enzyme operational and catalytic properties by directed mutagenesis. However, the structural-functional relationships explaining some desirable properties are not established yet and, therefore, their introduction was addressed by 'non-rational' directed evolution. Then, over 100 oxidative biotransformations were analyzed. Among them, it is noteworthy to mention the regio/stereo selective hydroxylation of long/short-chain alkanes (a chemically challenging reaction), epoxidation of alkenes, and production of hydroxy-fatty acids. Concerning aromatic oxygenations, the regioselective hydroxylation of flavonoids, and stereoselective hydroxylation/epoxidation of alkyl/alkenyl-benzenes were among the most remarkable reactions, together with enzymatic hydroxylation of benzene (as an alternative for harsh chemical process). Finally, peroxidases and peroxygenases also showed a potential as delignification biocatalysts and in the decolorization of contaminant dyes from textile industries.}, language = {en} } @misc{PiontekStrittmatterUllrichetal., author = {Piontek, Klaus and Strittmatter, Eric and Ullrich, Ren{\´e} and Gr{\"o}be, Glenn and Pecyna, Marek J. and Kluge, Martin and Scheibner, Katrin and Hofrichter, Martin and Plattner, Dietmar A.}, title = {Structural basis of substrate conversion in a new aromatic peroxygenase: cytochrome P450 functionality with benefits}, series = {The Journal of Biological Chemistry}, journal = {The Journal of Biological Chemistry}, number = {288}, issn = {1083-351X}, doi = {10.1074/jbc.M113.514521}, pages = {34767 -- 34776}, abstract = {Aromatic peroxygenases (APOs) represent a unique oxidoreductase sub-subclass of heme proteins with peroxygenase and peroxidase activity and were thus recently assigned a distinct EC classification (EC 1.11.2.1). They catalyze, inter alia, oxyfunctionalization reactions of aromatic and aliphatic hydrocarbons with remarkable regio- and stereoselectivities. When compared with cytochrome P450, APOs appear to be the choice enzymes for oxyfunctionalizations in organic synthesis due to their independence from a cellular environment and their greater chemical versatility. Here, the first two crystal structures of a heavily glycosylated fungal aromatic peroxygenase (AaeAPO) are described. They reveal different pH-dependent ligand binding modes. We model the fitting of various substrates in AaeAPO, illustrating the way the enzyme oxygenates polycyclic aromatic hydrocarbons. Spatial restrictions by a phenylalanine pentad in the active-site environment govern substrate specificity in AaeAPO.}, language = {en} }