19 results on '"Badenhorst, Christoffel P. S."'
Search Results
2. Engineering Regioselectivity of a P450 Monooxygenase Enables the Synthesis of Ursodeoxycholic Acid via 7β‐Hydroxylation of Lithocholic Acid.
- Author
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Grobe, Sascha, Badenhorst, Christoffel P. S., Bayer, Thomas, Hamnevik, Emil, Wu, Shuke, Grathwol, Christoph W., Link, Andreas, Koban, Sven, Brundiek, Henrike, Großjohann, Beatrice, and Bornscheuer, Uwe T.
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URSODEOXYCHOLIC acid , *WASTE products , *CYTOCHROME P-450 , *MOLECULAR docking , *ACIDS , *AMINO acid residues - Abstract
We engineered the cytochrome P450 monooxygenase CYP107D1 (OleP) from Streptomyces antibioticus for the stereo‐ and regioselective 7β‐hydroxylation of lithocholic acid (LCA) to yield ursodeoxycholic acid (UDCA). OleP was previously shown to hydroxylate testosterone at the 7β‐position but LCA is exclusively hydroxylated at the 6β‐position, forming murideoxycholic acid (MDCA). Structural and 3DM analysis, and molecular docking were used to identify amino acid residues F84, S240, and V291 as specificity‐determining residues. Alanine scanning identified S240A as a UDCA‐producing variant. A synthetic "small but smart" library based on these positions was screened using a colorimetric assay for UDCA. We identified a nearly perfectly regio‐ and stereoselective triple mutant (F84Q/S240A/V291G) that produces 10‐fold higher levels of UDCA than the S240A variant. This biocatalyst opens up new possibilities for the environmentally friendly synthesis of UDCA from the biological waste product LCA. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
3. Modifikation der Regioselektivität einer P450‐Monooxygenase ermöglicht die Synthese von Ursodeoxycholsäure durch die 7β‐Hydroxylierung von Lithocholsäure.
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Grobe, Sascha, Badenhorst, Christoffel P. S., Bayer, Thomas, Hamnevik, Emil, Wu, Shuke, Grathwol, Christoph W., Link, Andreas, Koban, Sven, Brundiek, Henrike, Großjohann, Beatrice, and Bornscheuer, Uwe T.
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STREPTOMYCES - Abstract
Wir haben die P450‐Monooxygenase CYP107D1 (OleP) aus Streptomyces antibioticus für die stereo‐ und regioselektive 7β‐Hydroxylierung von Lithocholsäure (LCS) zur Herstellung von Ursodeoxycholsäure (UDCS) durch "Protein‐Engineering" angepasst. OleP wurde zuvor für die Hydroxylierung von Testosteron an der 7β‐Position beschrieben, hydroxyliert jedoch LCS ausschließlich an der 6β‐Position, wodurch Murideoxycholsäure (MDCS) gebildet wird. Struktur‐ und 3DM‐Analysen, sowie molekulare Modellierungen wurden verwendet, um die Aminosäurereste F84, S240 und V291 als spezifitätsbestimmend zu identifizieren. Durch einen Alaninscan wurde S240A als UDCS‐produzierende Variante identifiziert. Basierend auf den identifizierten Positionen wurde eine synthetische "small but smart" Bibliothek durch die Verwendung eines farbbasierten Assays auf UDCS Produktion getestet. Hier konnte eine beinahe perfekte regio‐ und stereoselektive Dreifachvariante (F84Q/S240A/V291G) identifiziert werden, die UDCS in einer 10‐fach höheren Menge produziert als die S240A Variante. Der hergestellte Biokatalysator eröffnet neue Möglichkeiten zur umweltfreundlichen Synthese von UDCS aus dem Abfallprodukt LCS. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
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4. A Universal, Continuous Assay for SAM‐dependent Methyltransferases.
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Menke, Marian J., Schneider, Pascal, Badenhorst, Christoffel P. S., Kunzendorf, Andreas, Heinz, Florian, Dörr, Mark, Hayes, Martin A., and Bornscheuer, Uwe T.
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ESCHERICHIA coli , *METHYLTRANSFERASES , *HIGH throughput screening (Drug development) - Abstract
Enzyme‐catalyzed late‐stage functionalization (LSF), such as methylation of drug molecules and lead structures, enables direct access to more potent active pharmaceutical ingredients (API). S‐adenosyl‐l‐methionine‐dependent methyltransferases (MTs) can play a key role in the development of new APIs, as they catalyze the chemo‐ and regioselective methylation of O‐, N‐, S‐ and C‐atoms, being superior to traditional chemical routes. To identify suitable MTs, we developed a continuous fluorescence‐based, high‐throughput assay for SAM‐dependent methyltransferases, which facilitates screening using E. coli cell lysates. This assay involves two enzymatic steps for the conversion of S‐adenosyl‐l‐homocysteine into H2S to result in a selective fluorescence readout via reduction of an azidocoumarin sulfide probe. Investigation of two O‐MTs and an N‐MT confirmed that this assay is suitable for the determination of methyltransferase activity in E. coli cell lysates. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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5. Ein universeller, kontinuierlicher Assay für SAM‐abhängige Methyltransferasen.
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Menke, Marian J., Schneider, Pascal, Badenhorst, Christoffel P. S., Kunzendorf, Andreas, Heinz, Florian, Dörr, Mark, Hayes, Martin A., and Bornscheuer, Uwe T.
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ESCHERICHIA coli - Abstract
Die enzymkatalysierte Funktionalisierung (engl.: late‐stage functionalization (LSF)), wie z. B. die Methylierung von Arzneimittelmolekülen und Leitstrukturen, ermöglicht den direkten Zugang zu wirksameren pharmazeutischen Wirkstoffen (API). S‐Adenosyl‐l‐Methionin‐abhängige Methyltransferasen (MTs) können eine Schlüsselrolle bei der Entwicklung neuer Wirkstoffe spielen, da sie die chemo‐ und regioselektive Methylierung von O‐, N‐, S‐ und C‐Atomen katalysieren und damit den herkömmlichen chemischen Wegen überlegen sind. Um geeignete MTs zu identifizieren, haben wir einen kontinuierlichen fluoreszenzbasierten Hochdurchsatzassay für SAM‐abhängige Methyltransferasen entwickelt, der das Screening unter Verwendung von E. coli Zelllysaten erleichtert. Dieser Assay besteht aus zwei enzymatischen Schritten für die Umwandlung von S‐Adenosyl‐l‐Homocystein zu H2S, was zu einer selektiven Fluoreszenzmessung durch Reduktion eines Azidocoumarin‐Sulfidsensors führt. Die Untersuchung von zwei O‐MTs und einer N‐MT bestätigte, dass dieser Assay für die Bestimmung der Methyltransferase‐Aktivität in E. coli Zelllysaten geeignet ist. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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6. Cover Feature: Enzyme Kits to Facilitate the Integration of Biocatalysis into Organic Chemistry – First Aid for Synthetic Chemists (ChemCatChem 11/2022).
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Fessner, Nico D., Badenhorst, Christoffel P. S., and Bornscheuer, Uwe T.
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ORGANIC chemistry , *BIOCATALYSIS , *CHEMISTS , *ENZYMES , *ORGANIC synthesis - Abstract
Biocatalysis, early-stage functionalisation, enzyme kits, late-stage functionalisation, lead diversification Cover Feature: Enzyme Kits to Facilitate the Integration of Biocatalysis into Organic Chemistry - First Aid for Synthetic Chemists (ChemCatChem 11/2022) Keywords: biocatalysis; early-stage functionalisation; enzyme kits; late-stage functionalisation; lead diversification EN biocatalysis early-stage functionalisation enzyme kits late-stage functionalisation lead diversification 1 1 1 06/10/22 20220608 NES 220608 B The Cover Feature b illustrates how enzyme kits function as proficient and readily available "First Aid" for synthetic chemists, to simplify syntheses of complex molecules complementary to conventional chemical methods. [Extracted from the article]
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- 2022
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7. Strukturaufklärung einer metagenomischen Urethanase und Verbesserung des Hydrolyseprofils durch Protein Engineering.
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Bayer, Thomas, Palm, Gottfried J., Berndt, Leona, Meinert, Hannes, Branson, Yannick, Schmidt, Louis, Cziegler, Clemens, Somvilla, Ina, Zurr, Celine, Graf, Leonie G., Janke, Una, Badenhorst, Christoffel P. S., König, Stefanie, Delcea, Mihaela, Garscha, Ulrike, Wei, Ren, Lammers, Michael, and Bornscheuer, Uwe T.
- Abstract
Während Kunststoffe wie Polyethylenterephthalat (PET) bereits effizient durch die Aktivität von Hydrolasen abgebaut werden können, sind andere synthetische Polymere wie Polyurethane (PUs) und Polyamide (PAs) weitgehend resistent gegenüber einem biologischen Abbau. In dieser Studie lösten wir die erste Kristallstruktur der metagenomischen Urethanase UMG‐SP‐1, identifizierten hochflexible Loopregionen, die Reste des aktiven Zentrums enthalten, und untersuchten insgesamt 20 potenzielle Hotspots mittels Sättigungsmutagenese. Die durch Protein Engineering erzeugten Einzelmutanten wiesen eine fast 3‐ bzw. 8‐fach verbesserte Aktivität gegenüber hochstabilen N‐Arylurethan‐ und Amidbindungen auf. Darüber hinaus konnte die Freisetzung der entsprechenden Monomere aus einem thermoplastischen Polyester‐PU und einem PA (Nylon 6) durch die Aktivität einer einzigen, metagenomischen Urethanase nach kurzer Inkubationszeit nachgewiesen werden. Dadurch konnte das Hydrolyseprofil von UMG‐SP‐1 über die bekannten niedermolekularen Carbamate hinaus erweitert werden und erschließt so neue Möglichkeiten für den enzymatischen Abbau und das Recycling von Kunststoffen und Plastikabfällen. Damit unterstützt diese Studie Bemühungen um die Verbesserung einer Kreislaufwirtschaft für synthetische Polymere. [ABSTRACT FROM AUTHOR]
- Published
- 2024
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8. Structural Elucidation of a Metagenomic Urethanase and Its Engineering Towards Enhanced Hydrolysis Profiles.
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Bayer, Thomas, Palm, Gottfried J., Berndt, Leona, Meinert, Hannes, Branson, Yannick, Schmidt, Louis, Cziegler, Clemens, Somvilla, Ina, Zurr, Celine, Graf, Leonie G., Janke, Una, Badenhorst, Christoffel P. S., König, Stefanie, Delcea, Mihaela, Garscha, Ulrike, Wei, Ren, Lammers, Michael, and Bornscheuer, Uwe T.
- Abstract
While plastics like polyethylene terephthalate can already be degraded efficiently by the activity of hydrolases, other synthetic polymers like polyurethanes (PUs) and polyamides (PAs) largely resist biodegradation. In this study, we solved the first crystal structure of the metagenomic urethanase UMG‐SP‐1, identified highly flexible loop regions to comprise active site residues, and targeted a total of 20 potential hot spots by site‐saturation mutagenesis. Engineering campaigns yielded variants with single mutations, exhibiting almost 3‐ and 8‐fold improved activity against highly stable
N ‐aryl urethane and amide bonds, respectively. Furthermore, we demonstrated the release of the corresponding monomers from a thermoplastic polyester‐PU and a PA (nylon 6) by the activity of a single, metagenome‐derived urethanase after short incubation times. Thereby, we expanded the hydrolysis profile of UMG‐SP‐1 beyond the reported low‐molecular weight carbamates. Together, these findings promise advanced strategies for the bio‐based degradation and recycling of plastic materials and waste, aiding efforts to establish a circular economy for synthetic polymers. [ABSTRACT FROM AUTHOR]- Published
- 2024
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9. The diverse origins of circulating cell‐free DNA in the human body: a critical re‐evaluation of the literature.
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Aucamp, Janine, Bronkhorst, Abel J., Badenhorst, Christoffel P. S., and Pretorius, Piet J.
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DNA , *HUMAN body , *TUMORS , *GENETICS , *CANCER diagnosis , *GENE therapy , *IMMUNOTHERAPY , *THERAPEUTICS - Abstract
ABSTRACT: Since the detection of cell‐free DNA (cfDNA) in human plasma in 1948, it has been investigated as a non‐invasive screening tool for many diseases, especially solid tumours and foetal genetic abnormalities. However, to date our lack of knowledge regarding the origin and purpose of cfDNA in a physiological environment has limited its use to more obvious diagnostics, neglecting, for example, its potential utility in the identification of predisposition to disease, earlier detection of cancers, and lifestyle‐induced epigenetic changes. Moreover, the concept or mechanism of cfDNA could also have potential therapeutic uses such as in immuno‐ or gene therapy. This review presents an extensive compilation of the putative origins of cfDNA and then contrasts the contributions of cellular breakdown processes with active mechanisms for the release of cfDNA into the extracellular environment. The involvement of cfDNA derived from both cellular breakdown and active release in lateral information transfer is also discussed. We hope to encourage researchers to adopt a more holistic view of cfDNA research, taking into account all the biological pathways in which cfDNA is involved, and to give serious consideration to the integration of in vitro and in vivo research. We also wish to encourage researchers not to limit their focus to the apoptotic or necrotic fraction of cfDNA, but to investigate the intercellular messaging capabilities of the actively released fraction of cfDNA and to study the role of cfDNA in pathogenesis. [ABSTRACT FROM AUTHOR]
- Published
- 2018
- Full Text
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10. Urethanases for the Enzymatic Hydrolysis of Low Molecular Weight Carbamates and the Recycling of Polyurethanes.
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Branson, Yannick, Söltl, Simone, Buchmann, Carolin, Wei, Ren, Schaffert, Lena, Badenhorst, Christoffel P. S., Reisky, Lukas, Jäger, Gernot, and Bornscheuer, Uwe T.
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MOLECULAR weights , *CARBAMATES , *HYDROLYSIS , *URETHANE , *GLYCOLYSIS , *PLASTICS , *POLYURETHANE elastomers , *URETHANE foam - Abstract
Enzymatic degradation and recycling can reduce the environmental impact of plastics. Despite decades of research, no enzymes for the efficient hydrolysis of polyurethanes have been reported. Whereas the hydrolysis of the ester bonds in polyester‐polyurethanes by cutinases is known, the urethane bonds in polyether‐polyurethanes have remained inaccessible to biocatalytic hydrolysis. Here we report the discovery of urethanases from a metagenome library constructed from soil that had been exposed to polyurethane waste for many years. We then demonstrate the use of a urethanase in a chemoenzymatic process for polyurethane foam recycling. The urethanase hydrolyses low molecular weight dicarbamates resulting from chemical glycolysis of polyether‐polyurethane foam, making this strategy broadly applicable to diverse polyether‐polyurethane wastes. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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11. Urethanasen für die enzymatische Hydrolyse niedermolekularer Carbamate und das Recycling von Polyurethanen.
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Branson, Yannick, Söltl, Simone, Buchmann, Carolin, Wei, Ren, Schaffert, Lena, Badenhorst, Christoffel P. S., Reisky, Lukas, Jäger, Gernot, and Bornscheuer, Uwe T.
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WASTE recycling , *ENZYMES , *POLYETHERS , *POLYESTERS - Abstract
Enzymatischer Abbau und Recycling können die Umweltbelastung durch Plastik reduzieren. Trotz jahrzehntelanger Forschung wurde bisher nicht über Enzyme für die effiziente Hydrolyse von Polyurethanen berichtet. Während die Hydrolyse von Esterbindungen in Polyester‐Polyurethanen durch Cutinasen bekannt ist, blieben die Urethanbindungen in Polyether‐Polyurethanen für biokatalytische Hydrolyse bislang unzugänglich. Wir berichten hier über die Entdeckung von Urethanasen in einer Metagenombibliothek, erstellt aus einer Bodenprobe, welche Polyurethanabfall für viele Jahre ausgesetzt war. Wir demonstrieren weiterhin die Anwendung einer Urethanase in einem chemo‐enzymatischen Prozess zum Polyurethan‐Recycling. Diese Urethanase hydrolysiert niedermolekulare Dicarbamate, welche aus der chemischen Glykolyse von Polyether‐Polyurethanschaum entstehen, was diese Strategie breit anwendbar auf diverse Polyether‐Polyurethan Abfälle macht. [ABSTRACT FROM AUTHOR]
- Published
- 2023
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12. Engineering and evaluation of thermostable IsPETase variants for PET degradation.
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Brott, Stefan, Pfaff, Lara, Schuricht, Josephine, Schwarz, Jan‐Niklas, Böttcher, Dominique, Badenhorst, Christoffel P. S., Wei, Ren, and Bornscheuer, Uwe T.
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CHEMICAL recycling , *GLASS transition temperature , *POLYETHYLENE terephthalate , *MELTING points - Abstract
Polyethylene terephthalate (PET) is a mass‐produced petroleum‐based synthetic polymer. Enzymatic PET degradation using, for example, Ideonella sakaiensis PETase (IsPETase) can be a more environmentally friendly and energy‐saving alternative to the chemical recycling of PET. However, IsPETase is a mesophilic enzyme with an optimal reaction temperature lower than the glass transition temperature (Tg) of PET, where the amorphous polymers can be readily accessed for enzymatic breakdown. In this study, we used error‐prone PCR to generate a mutant library based on a thermostable triple mutant (TM) of IsPETase. The library was screened against the commercially available polyester‐polyurethane Impranil DLN W 50 for more thermostable IsPETase variants, yielding four variants with higher melting points. The most promising IsPETaseTMK95N/F201I variant had a 5.0°C higher melting point than IsPETaseTM. Although this variant showed a slightly lower activity on PET at lower incubation temperatures, its increased thermostability makes it a more active PET hydrolase at higher reaction temperatures up to 60°C. Several other variants were compared and combined with selected previously published IsPETase mutants in terms of thermostability and hydrolytic activity against PET nanoparticles and amorphous PET films. Our findings indicate that thermostability is one of the most important characteristics of an effective PET hydrolase. [ABSTRACT FROM AUTHOR]
- Published
- 2022
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13. Discovery and Design of Family VIII Carboxylesterases as Highly Efficient Acyltransferases.
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Müller, Henrik, Godehard, Simon P., Palm, Gottfried J., Berndt, Leona, Badenhorst, Christoffel P. S., Becker, Ann‐Kristin, Lammers, Michael, and Bornscheuer, Uwe T.
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ACYLTRANSFERASES , *CARBOXYLESTERASES , *DRUG synthesis , *HYDROLASES , *CRYSTAL structure , *ACETYLTRANSFERASES - Abstract
Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activity‐based screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4‐chlorobenzoate and 4‐(2‐aminoethyl)morpholine (ca. 20 % conversion). We solved the crystal structure of EstCE1 and detailed structure–function analysis revealed a three‐amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a "hydrolase" into an "acyltransferase". [ABSTRACT FROM AUTHOR]
- Published
- 2021
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14. Entdeckung und Design promiskuitiver Acyltransferase‐Aktivität in Carboxylesterasen der Familie VIII.
- Author
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Müller, Henrik, Godehard, Simon P., Palm, Gottfried J., Berndt, Leona, Badenhorst, Christoffel P. S., Becker, Ann‐Kristin, Lammers, Michael, and Bornscheuer, Uwe T.
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DESIGN , *ACYLTRANSFERASES - Abstract
Promiskuitive Acyltransferase‐Aktivität ist die Eigenschaft mancher Hydrolasen, in wässriger Lösung Acyltransfer statt Hydrolyse zu katalysieren. Niedrige Enantioselektivität bzw. Effizienz, signifikante Produkthydrolyse und das begrenzte Substratspektrum bekannter Acyltransferasen stellen erhebliche Nachteile für industrielle Anwendungen dar. Durch aktivitätsbasiertes Screening von Hydrolasen konnte die Carboxylesterase EstCE1 aus der Familie VIII als beispiellos effiziente Acyltransferase identifiziert werden. EstCE1 kann die irreversible Amidierung und Carbamoylierung von Aminen in Wasser katalysieren, wie hier am Beispiel der Synthese des Wirkstoffs Moclobemid gezeigt. Detaillierte Struktur‐Funktions‐Analysen basierend auf der hier gelösten Kristallstruktur von EstCE1 identifizierten ein Sequenz‐Motiv, das sich als bedeutsam für die promiskuitive Acyltransferase‐Aktivität herausstellte. Die Übertragung dieses Motivs führte zur Umwandlung einer "Hydrolase" in eine "Acyltransferase". [ABSTRACT FROM AUTHOR]
- Published
- 2021
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- View/download PDF
15. Directed Evolution of a Halide Methyltransferase Enables Biocatalytic Synthesis of Diverse SAM Analogs.
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Tang, Qingyun, Grathwol, Christoph W., Aslan‐Üzel, Aşkın S., Wu, Shuke, Link, Andreas, Pavlidis, Ioannis V., Badenhorst, Christoffel P. S., and Bornscheuer, Uwe T.
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BIOCATALYSIS , *HALIDES , *METHYL iodide , *NUCLEAR magnetic resonance spectroscopy , *ARABIDOPSIS thaliana , *ETHYLATION - Abstract
Biocatalytic alkylations are important reactions to obtain chemo‐, regio‐ and stereoselectively alkylated compounds. This can be achieved using S‐adenosyl‐l‐methionine (SAM)‐dependent methyltransferases and SAM analogs. It was recently shown that a halide methyltransferase (HMT) from Chloracidobacterium thermophilum can synthesize SAM from SAH and methyl iodide. We developed an iodide‐based assay for the directed evolution of an HMT from Arabidopsis thaliana and used it to identify a V140T variant that can also accept ethyl‐, propyl‐, and allyl iodide to produce the corresponding SAM analogs (90, 50, and 70 % conversion of 15 mg SAH). The V140T AtHMT was used in one‐pot cascades with O‐methyltransferases (IeOMT or COMT) to achieve the regioselective ethylation of luteolin and allylation of 3,4‐dihydroxybenzaldehyde. While a cascade for the propylation of 3,4‐dihydroxybenzaldehyde gave low conversion, the propyl‐SAH intermediate could be confirmed by NMR spectroscopy. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
16. Die gerichtete Evolution einer Halogenid‐Methyltransferase erlaubt die biokatalytische Synthese diverser SAM‐Analoga.
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Tang, Qingyun, Grathwol, Christoph W., Aslan‐Üzel, Aşkın S., Wu, Shuke, Link, Andreas, Pavlidis, Ioannis V., Badenhorst, Christoffel P. S., and Bornscheuer, Uwe T.
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LUTEOLIN - Abstract
Biokatalytische Alkylierungen sind wichtige Reaktionen, um chemo‐, regio‐ und stereoselektiv alkylierte Verbindungen zu erhalten. Dies kann mit S‐Adenosyl‐l‐methionin (SAM)‐abhängigen Methyltransferasen und SAM‐Analoga erreicht werden. Kürzlich wurde gezeigt, dass eine Halogenid‐Methyltransferase (HMT) aus Chloracidobacterium thermophilum SAM ausgehend von SAH und Methyliodid herstellen kann. Wir entwickelten einen Iodid‐basierten Assay für die gerichtete Evolution einer HMT aus Arabidopsis thaliana und nutzten diesen, um eine V140T‐Variante zu identifizieren, die auch Ethyl‐, Propyl‐, und Allyliodid akzeptiert, um die entsprechenden SAM‐Analoga herzustellen (90, 50 und 70 % Umsatz ausgehend von 15 mg SAH). Die V140T‐AtHMT wurde in einer Eintopfkaskade mit O‐Methyltransferasen (IeOMT und COMT) eingesetzt, um die regioselektive Alkylierung von Luteolin und 3,4‐Dihydroxybenzaldehyd zu erzielen. Obwohl eine Kaskade zur Propylierung von 3,4‐Dihydroxybenzaldehyd niedrige Umsätze ergab, konnte das Propyl‐SAH‐Intermediat durch NMR‐Spektroskopie bestätigt werden. [ABSTRACT FROM AUTHOR]
- Published
- 2021
- Full Text
- View/download PDF
17. Sequence‐Based Prediction of Promiscuous Acyltransferase Activity in Hydrolases.
- Author
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Müller, Henrik, Becker, Ann‐Kristin, Palm, Gottfried J., Berndt, Leona, Badenhorst, Christoffel P. S., Godehard, Simon P., Reisky, Lukas, Lammers, Michael, and Bornscheuer, Uwe T.
- Subjects
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HYDROLASES , *ACYLTRANSFERASES , *FORECASTING , *AMINO acid sequence , *ESTERASES - Abstract
Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate‐binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
18. Sequence‐Based Prediction of Promiscuous Acyltransferase Activity in Hydrolases.
- Author
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Müller, Henrik, Becker, Ann‐Kristin, Palm, Gottfried J., Berndt, Leona, Badenhorst, Christoffel P. S., Godehard, Simon P., Reisky, Lukas, Lammers, Michael, and Bornscheuer, Uwe T.
- Subjects
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HYDROLASES , *ACYLTRANSFERASES , *FORECASTING , *AMINO acid sequence , *ESTERASES - Abstract
Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate‐binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
19. An Ultrasensitive Fluorescence Assay for the Detection of Halides and Enzymatic Dehalogenation.
- Author
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Aslan‐Üzel, Aşkın S., Beier, Andy, Kovář, David, Cziegler, Clemens, Padhi, Santosh K., Schuiten, Eva D., Dörr, Mark, Böttcher, Dominique, Hollmann, Frank, Rudroff, Florian, Mihovilovic, Marko D., Buryška, Tomáš, Damborský, Jiří, Prokop, Zbyněk, Badenhorst, Christoffel P. S., and Bornscheuer, Uwe T.
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DEHALOGENATION , *HALIDES , *FLUORESCENCE , *BROMIDE ions , *DEHALOGENASES , *FLUORESCEIN - Abstract
Halide assays are important for the study of enzymatic dehalogenation, a topic of great industrial and scientific importance. Here we describe the development of a very sensitive halide assay that can detect less than a picomole of bromide ions, making it very useful for quantifying enzymatic dehalogenation products. Halides are oxidised under mild conditions using the vanadium‐dependent chloroperoxidase from Curvularia inaequalis, forming hypohalous acids that are detected using aminophenyl fluorescein. The assay is up to three orders of magnitude more sensitive than currently available alternatives, with detection limits of 20 nM for bromide and 1 μM for chloride and iodide. We demonstrate that the assay can be used to determine specific activities of dehalogenases and validate this by comparison to a well‐established GC‐MS method. This new assay will facilitate the identification and characterisation of novel dehalogenases and may also be of interest to those studying other halide‐producing enzymes. [ABSTRACT FROM AUTHOR]
- Published
- 2020
- Full Text
- View/download PDF
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