TY - GEN A1 - Kluge, Martin A1 - Ullrich, René A1 - Dolge, Christoph A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Hydroxylation of naphthalene by aromatic peroxygenase from Agrocybe aegerita proceeds via oxygen transfer from H2O2 and intermediary epoxidation T2 - Applied Microbiology and Biotechnology N2 - 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. KW - Peroxidase KW - Oxygenation KW - Hydroxylation KW - P450 KW - Naphthol Y1 - 2009 UR - http://link.springer.com/article/10.1007%2Fs00253-008-1704-y U6 - https://doi.org/10.1007/s00253-008-1704-y SN - 0175-7598 VL - 81 IS - 6 SP - 1071 EP - 1076 ER - TY - GEN A1 - Kluge, Martin A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Spectrophotometric assay for detection of aromatic hydroxylation catalyzed by fungal haloperoxidase–peroxygenase T2 - Applied Microbiology and Biotechnology N2 - 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. KW - Hydroxylation KW - Peroxygenase Y1 - 2007 UR - http://link.springer.com/article/10.1007%2Fs00253-007-0942-8 U6 - https://doi.org/10.1007/s00253-007-0942-8 SN - 1432-0614 VL - 75 IS - 6 SP - 1473 EP - 1478 ER - TY - GEN A1 - Poraj-Kobielska, Marzena A1 - Kinne, Matthias A1 - Ullrich, René A1 - Scheibner, Katrin A1 - Kayser, Gernot A1 - Hammel, Kenneth E. A1 - Hofrichter, Martin T1 - Preparation of human drug metabolites using fungal peroxygenases T2 - Biochemical Pharmacology N2 - 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. KW - Peroxidase KW - Peroxygenation KW - Hydroxylation KW - O-Dealkylation KW - N-Dealkylation KW - Cytochrome P450 Y1 - 2011 UR - http://www.sciencedirect.com/science/article/pii/S0006295211004035 U6 - https://doi.org/10.1016/j.bcp.2011.06.020 SN - 1873-2968 VL - 82 IS - 7 SP - 789 EP - 796 ER - TY - GEN A1 - Kinne, Matthias A1 - Poraj-Kobielska, Marzena A1 - Aranda, Elisabet A1 - Ullrich, René A1 - Hammel, Kenneth E. A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Regioselective preparation of 5-hydroxypropranolol and 4′-hydroxydiclofenac with a fungal peroxygenase T2 - Bioorganic & Medicinal Chemistry Letters N2 - 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. KW - Peroxidase KW - Peroxygenase KW - Oxygenase KW - Cytochrome P450 KW - Hydroxylation KW - 5-Hydroxypropranolol KW - Propranolol KW - 4′-Hydroxydiclofenac KW - Diclofenac KW - Ascorbic acid Y1 - 2009 UR - http://www.sciencedirect.com/science/article/pii/S0960894X09005071 U6 - https://doi.org/10.1016/j.bmcl.2009.04.015 SN - 1464-3405 VL - 19 IS - 11 SP - 3085 EP - 3087 ER - TY - GEN A1 - Kinne, Matthias A1 - Ullrich, René A1 - Hammel, Kenneth E. A1 - Scheibner, Katrin A1 - Hofrichter, Martin T1 - Regioselective preparation of (R)-2-(4-Hydroxyphenoxy)propionic acid with a fungal peroxygenase T2 - Tetrahedron Letters N2 - 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. KW - Peroxidase KW - Peroxygenase KW - Oxygenase KW - Cytochrome P450 KW - Hydroxylation KW - 2-(4-Hydroxyphenoxy)propionic acid KW - Ascorbic acid Y1 - 2008 UR - http://www.sciencedirect.com/science/article/pii/S0040403908014421 U6 - https://doi.org/10.1016/j.tetlet.2008.07.152 SN - 1873-3581 VL - 49 IS - 41 SP - 5950 EP - 5953 ER -