Pyridine as novel substrate for regioselective oxygenation with aromatic peroxygenase from Agrocybe aegerita

AbstractAgrocybe aegerita peroxidase (AaP) is a versatile extracellular biocatalyst that can oxygenate aromatic compounds. Here, we report on the selective oxidation of pyridine (PY) yielding pyridine N-oxide as sole product. Using H218O2 as co-substrate, the origin of oxygen was confirmed to be the peroxide. Therefore, AaP can be regarded as a true peroxygenase transferring one oxygen atom from peroxide to the substrate. To our best knowledge, there are only two types of enzymes oxidizing PY at the nitrogen: bacterial methane monooxygenase and a few P450 monooxygenases. AaP is the first extracellular enzyme and the first peroxidase that catalyzes this reaction, and it converted also substituted PYs into the corresponding N-oxides

How experiments and molecular simulations can help understand selective C25-hydroxylation of vitamin D by fungal peroxygenases

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Molecular determinants for selective C 25-hydroxylation of vitamins D 2 and D 3 by fungal peroxygenases

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 ReviewedPostprint (author's final draft

Protein Kinase C Delta (PKCδ) Affects Proliferation of Insulin-Secreting Cells by Promoting Nuclear Extrusion of the Cell Cycle Inhibitor p21Cip1/WAF1

BACKGROUND:High fat diet-induced hyperglycemia and palmitate-stimulated apoptosis was prevented by specific inhibition of protein kinase C delta (PKCδ) in β-cells. To understand the role of PKCδ in more detail the impact of changes in PKCδ activity on proliferation and survival of insulin-secreting cells was analyzed under stress-free conditions. METHODOLOGY AND PRINCIPAL FINDINGS:Using genetic and pharmacological approaches, the effect of reduced and increased PKCδ activity on proliferation, apoptosis and cell cycle regulation of insulin secreting cells was examined. Proteins were analyzed by Western blotting and by confocal laser scanning microscopy. Increased expression of wild type PKCδ (PKCδWT) significantly stimulated proliferation of INS-1E cells with concomitant reduced expression and cytosolic retraction of the cell cycle inhibitor p21(Cip1/WAF1). This nuclear extrusion was mediated by PKCδ-dependent phosphorylation of p21(Cip1/WAF1) at Ser146. In kinase dead PKCδ (PKCδKN) overexpressing cells and after inhibition of endogenous PKCδ activity by rottlerin or RNA interference phosphorylation of p21(Cip1/WAF1) was reduced, which favored its nuclear accumulation and apoptotic cell death of INS-1E cells. Human and mouse islet cells express p21(Cip1/WAF1) with strong nuclear accumulation, while in islet cells of PKCδWT transgenic mice the inhibitor resides cytosolic. CONCLUSIONS AND SIGNIFICANCE:These observations disclose PKCδ as negative regulator of p21(Cip1/WAF1), which facilitates proliferation of insulin secreting cells under stress-free conditions and suggest that additional stress-induced changes push PKCδ into its known pro-apoptotic role

Wissenschaft als Beruf: Wissenschaftsforschung Jahrbuch 2020

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Synthesis of Indigo-Dyes from Indole Derivatives by Unspecific Peroxygenases and Their Application for In-Situ Dyeing

Tyrian purple (also known as royal or imperial purple) is the oldest known commercial pigment and still one of the most expensive dyes, often associated with the wardrobes of clergy and royalty. It is a brominated derivative of indigo, a natural dye that has been used since 4000 BC. Moreover, just recently, the therapeutic value of indigoids for the treatment of several disorders was discovered. The manufacturing of indigo derivatives by the existing chemical routes has become increasingly uninteresting due to the use of aggressive reagents, expensive starting materials and high-energy costs. Thus, both dyestuff manufacturers and the pharmaceutical industry are interested in the development of gentle preparation methods of indigoids from simple precursors. Here, we describe a simple enzymatic method for the one-step synthesis of Tyrian purple and other indigo derivatives with fungal peroxygenases (UPO, EC 1.11.2.1). The reaction does not require complex co-substrates and works well in phosphate buffers with H2O2 (<0.1 wt%) and less than 5% (v/v) acetonitrile as co-solvent. We demonstrate the scaling up of the reaction to 10 Liters and established thereupon an environmentally friendly combined synthesis and in-situ dyeing process, further simplifying the manufacturing of vat-dyed fabrics. Eventually, we screened a number of halogen-substituted indoles in the search for novel indigo derivatives, which may be of interest for pharmaceutical and/or dyeing purposes

Zbiorowisko grzybów na cmentarzu „Gottesacker” w Herrnhut (Górne Łużyce, Saksonia)

„Gottesacker“ v Herrnhutu (Horní Lužice, Sasko) je hřbitovem téměř 300 let. G. Zschieschang intenzivně mapoval své houbové společenství od 60. let 20. století. Spojením jeho nálezů a nálezů jiných mykologů zde můžeme analyzovat mykologická data, která zachycují asi 55 let. Biologickou rozmanitost hub posuzujeme se zvláštním důrazem na druhy CHEGD (Clavariaceae-Hygrocybe-Entoloma-Geoglossaceae-Dermoloma). Tyto druhy se používají v soupisech ke klasifikaci a k posouzení toho, zda si travnaté porosty zachovávají svou hodnotu. Podle námi určeného profilu CHEGD lze „Gottesacker“ zařadit k travním porostům mezinárodního významu. Dále uvádíme makroskopické a mikroskopické charakteristiky dvou vzácných druhů CHEGD: Entoloma brunneosericeum a Clavaria messapica. Jedná se o první nálezy obou druhů v Sasku (prvního jmenovaného dokonce v celém Německu).Der „Gottesacker“ in Herrnhut (Oberlausitz, Sachsen) ist seit fast 300 Jahren ein Friedhof. G. Zschieschang hatte seine Pilzgemeinschaft seit den 1960er Jahren intensiv kartiert. Indem wir seine Erkenntnisse und die anderer Mykologen zusammenführen, können wir hier mykologische Daten analysieren, die etwa 55 Jahre umfassen. Dabei behandeln wir die pilzliche Biodiversität mit besonderem Augenmerk auf den CHEGD-Arten (Clavariaceae-Hygrocybe-Entoloma-Geoglossaceae-Dermoloma). Diese Arten werden im Zuge von Inventarisierungen herangezogen, um den Erhaltungswert von Grünland zu klassifizieren und zu bewerten. Entsprechend dem von uns ermittelten CHEGD-Profil ist der "Gottesacker" als Grünland von internationaler Bedeutung einzustufen. Darüber hinaus präsentieren wir makroskopische und mikroskopische Charakteristika von zwei seltenen CHEGD-Arten: Entoloma brunneosericeum und Clavaria messapica. Dies sind die Erstnachweise beider Arten für Deutschland bzw. Sachsen.The Gottesacker (“God’s acre”) in Herrnhut (Upper Lusatia, Saxony) has been a graveyard for almost 300 years. G. Zschieschang has mapped its fungal community since the 1960ies. Combining these findings and those of other mycologists, mycological data that cover about 55 years are reported here. In this context, we discuss the fungal biodiversity with special emphasis on CHEGD species (Clavariaceae-Hygrocybe-Entoloma-Geoglossaceae-Dermoloma). These species are used to classify and assess the conservation value of grasslands by monitoring their fungal communities. According to the determined CHEGD profile, the Gottesacker graveyard is ranked as a grassland of international importance. In addition, we present macroscopic and microscopic characteristics of two rare CHEGD species: Entoloma brunneosericeum and Clavaria messapica. These are the first records of both species for Germany and Saxony, respectively.„Gottesacker“ w mieście Herrnhut (Górne Łużyce, Saksonia) to cmentarz istniejący od ponad 300 lat. Lokalne zbiorowiska grzybów intensywnie od lat 60. XX wieku badał G. Zschieschang. Łącząc jego obserwacje z obserwacjami innych mykologów, możemy analizować dane mykologiczne obejmujące okres około 55 lat. Różnorodność biologiczną grzybów oceniamy ze szczególnym uwzględnieniem gatunków CHEGD (Clavariaceae-Hygrocybe-Entoloma-Geoglossaceae-Dermoloma). Te gatunki stosowane są w dokumentacji do celów klasyfikacji i oceny, czy użytki zielone zachowują swoją wartość. Na podstawie określonego przez nas profilu CHEGD, „Gottesacker“ można zaliczyć do użytków zielonych o znaczeniu międzynarodowym. Ponadto przedstawiamy makroskopowe i mikroskopowe cechy dwóch rzadkich gatunków CHEGD: Entoloma brunneosericeum i Clavaria messapica. Gatunki te po raz pierwszy zaobserwowano w Saksonii (pierwszy z nich nawet w skali całych Niemiec)

Regioselective oxygenation of fatty acids, fatty alcohols and other aliphatic compounds by a basidiomycete heme-thiolate peroxidase

Reaction of fatty acids, fatty alcohols, alkanes, sterols, sterol esters and triglycerides with the so-called aromatic peroxygenase from Agrocybe aegerita was investigated using GC–MS. Regioselective hydroxylation of C12–C20 saturated/unsaturated fatty acids was observed at the ω-1 and ω-2 positions (except myristoleic acid only forming the ω-2 derivative). Minor hydroxylation at ω and ω-3 to ω-5 positions was also observed. Further oxidized products were detected, including keto, dihydroxylated, ketohydroxy and dicarboxylic fatty acids. Fatty alcohols also yielded hydroxy or keto derivatives of the corresponding fatty acid. Finally, alkanes gave, in addition to alcohols at positions 2 or 3, dihydroxylated derivatives at both sides of the molecule; and sterols showed side-chain hydroxylation. No derivatives were found for fatty acids esterified with sterols or forming triglycerides, but methyl esters were ω-1 or ω-2 hydroxylated. Reactions using H218O2 established that peroxide is the source of the oxygen introduced in aliphatic hydroxylations. These studies also indicated that oxidation of alcohols to carbonyl and carboxyl groups is produced by successive hydroxylations combined with one dehydration step. We conclude that the A. aegerita peroxygenase not only oxidizes aromatic compounds but also catalyzes the stepwise oxidation of aliphatic compounds by hydrogen peroxide, with different hydroxylated intermediates.This study was funded by the PEROXICATS (KBBE-2010-4-265397) and BIORENEW (NMP2-CT-2006-026456) EU-projects, and the ELLE (AGL2008-00709) and RAPERO (BIO2008-01533) Spanish MICINN projects co-financed by FEDER funds.Peer reviewe

Enzymatic Preparation of 2,5-Furandicarboxylic Acid (FDCA)—A Substitute of Terephthalic Acid—By the Joined Action of Three Fungal Enzymes

Enzymatic oxidation of 5-hydroxymethylfurfural (HMF) and its oxidized derivatives was studied using three fungal enzymes: wild-type aryl alcohol oxidase (AAO) from three fungal species, wild-type peroxygenase from Agrocybe aegerita (AaeUPO), and recombinant galactose oxidase (GAO). The effect of pH on different reaction steps was evaluated and apparent kinetic data (Michaelis-Menten constants, turnover numbers, specific constants) were calculated for different enzyme-substrate ratios and enzyme combinations. Finally, the target product, 2,5-furandicarboxylic acid (FDCA), was prepared in a multi-enzyme cascade reaction combining three fungal oxidoreductases at micro-scale. Furthermore, an oxidase-like reaction is proposed for heme-containing peroxidases, such as UPO, horseradish peroxidase, or catalase, causing the conversion of 5-formyl-2-furancarboxylic acid into FDCA in the absence of exogenous hydrogen peroxide