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Cbic.202000100Taverne Delft University of Technology A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications Phonbuppha, Jittima; Tinikul, Ruchanok; Wongnate, Thanyaporn; Intasian, Pattarawan; Hollmann, Frank; Paul, Caroline E.; Chaiyen, Pimchai DOI 10.1002/cbic.202000100 Publication date 2020 Document Version Final published version Published in ChemBioChem Citation (APA) Phonbuppha, J., Tinikul, R., Wongnate, T., Intasian, P., Hollmann, F., Paul, C. E., & Chaiyen, P. (2020). A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications. ChemBioChem, 21(14), 2073-2079. https://doi.org/10.1002/cbic.202000100 Important note To cite this publication, please use the final published version (if applicable). Please check the document version above. Copyright Other than for strictly personal use, it is not permitted to download, forward or distribute the text or part of it, without the consent of the author(s) and/or copyright holder(s), unless the work is under an open content license such as Creative Commons. Takedown policy Please contact us and provide details if you believe this document breaches copyrights. We will remove access to the work immediately and investigate your claim. This work is downloaded from Delft University of Technology. For technical reasons the number of authors shown on this cover page is limited to a maximum of 10. Green Open Access added to TU Delft Institutional Repository ‘You share, we take care!’ – Taverne project https://www.openaccess.nl/en/you-share-we-take-care Otherwise as indicated in the copyright section: the publisher is the copyright holder of this work and the author uses the Dutch legislation to make this work public. Full Papers ChemBioChem doi.org/10.1002/cbic.202000100 1 2 3 A Minimized Chemoenzymatic Cascade for Bacterial 4 5 Luciferase in Bioreporter Applications 6 [a] [b] [a] [a] 7 Jittima Phonbuppha, Ruchanok Tinikul, Thanyaporn Wongnate, Pattarawan Intasian, [c] [c] [a, b] 8 Frank Hollmann, Caroline E. Paul, and Pimchai Chaiyen* 9 10 11 Bacterial luciferase (Lux) catalyzes a bioluminescence reaction nicotinamide (BNAH) was used in place of the flavin reductase 12 by using long-chain aldehyde, reduced flavin and molecular reaction in the Lux system. The results showed that the 13 oxygen as substrates. The reaction can be applied in reporter minimized cascade reaction can be applied to monitor bio- 14 gene systems for biomolecular detection in both prokaryotic luminescence of the Lux reporter in eukaryotic cells effectively, 15 and eukaryotic organisms. Because reduced flavin is unstable and that it can achieve higher efficiencies than the system with 16 under aerobic conditions, another enzyme, flavin reductase, is flavin reductase. This development is useful for future applica- 17 needed to supply reduced flavin to the Lux-catalyzed reaction. tions as high-throughput detection tools for drug screening 18 To create a minimized cascade for Lux that would have greater applications. 19 ease of use, a chemoenzymatic reaction with a biomimetic 20 21 Introduction 22 23 Two component flavin-dependent monooxygenases are enzy- 24 matic systems composed of a monooxygenase that catalyzes 25 the main oxygenation reaction and a flavin reductase that 26 supplies reduced flavin as a substrate for the monooxygenase 27 component. These enzymes are useful for various applications 28 as they can catalyze chemo-, regio- and enantio-selective 29 monooxygenation reactions. Previous examples have shown 30 the application of these enzymes in biocatalysis applications for 31 the production of active pharmaceutical ingredients and other 32 useful chemicals,[1] as well as for bioremediation and biodetec- 33 tion applications for toxic compounds.[2] Successful application Scheme 1. A general catalytic cycle of two-component flavin monooxyge- 34 nases. of these enzymes requires well-coordinated tandem enzymatic 35 reactions of the flavin reductase and the monooxygenase. 36 Previous investigations have shown that the reduced flavin can 37 be transferred to the monooxygenase via diffusion.[3] Therefore, nases to react with molecular oxygen to yield C4a-(hydro) 38 the steady-state levels of reduced flavin and the rate at which it peroxyflavin which is the key intermediate of all flavin-depend- 39 is produced must be carefully optimized in order to avoid ent monooxygenases.[5] The C4a-(hydro)peroxyflavin intermedi- 40 wasteful production of reduced flavin, which can be rendered ate can react with different substrates depending on the 41 unusable by oxidation by molecular oxygen to generate H O catalytic properties of the specific enzymes to yield oxygenated 42 2 2 (Scheme 1).[4] The reduced flavin is used by the monooxyge- products.[6] 43 Bacterial luciferase (Lux) is a two-component flavin-depend- 44 ent monooxygenase that catalyzes the oxidation of long chain 45 [a] J. Phonbuppha, Dr. T. Wongnate, P. Intasian, Prof. P. Chaiyen aldehydes using reduced FMN (FMNHÀ ) and molecular oxygen 46 School of Biomolecular Science and Engineering (BSE) Vidyasirimedhi Institute of Science and Technology (VISTEC) as co-substrates to yield carboxylic acid, oxidized flavin and 47 555 Moo 1 Payupnai, Wangchan, Rayong, 21210 (Thailand) water with concomitant emission of blue-green light at 48 E-mail: [email protected] 490 nm.[6] Lux is encoded by the luxAB genes in the lux operon 49 [b] Dr. R. Tinikul, Prof. P. Chaiyen Department of Biochemistry and Center for Excellence in Protein and of luminous bacteria which also contain the luxCDE genes 50 Enzyme Technology, Faculty of Science encoding for a fatty acid reductase complex that catalyzes the 51 Mahidol University conversion of fatty acid to aldehyde.[7] Some luminous bacterial 52 272 Rama VI Road, Ratchathewi, Bangkok, 10400 (Thailand) [c] Prof. F. Hollmann, Dr. C. E. Paul strains also contain the luxG gene which encodes for a flavin 53 Department of Biotechnology reductase that catalyzes the production of reduced flavin.[8] 54 Delft University of Technology Based on the ability to generate bioluminescence, Lux has been 55 Van der Maasweg 9, 2629 HZ Delft (The Netherlands) used as a reporter gene in whole-cell bacterial biosensors for 56 This article is part of a joint Special Collection dedicated to the Biotrans 2019 symposium. To view the complete collection, visit our homepage monitoring levels of specific analytes[9] related to oxidative 57 ChemBioChem 2020, 21, 1–8 1 © 2020 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim These are not the final page numbers! �� Wiley VCH Dienstag, 14.04.2020 2099 / 164435 [S. 1/8] 1 Full Papers ChemBioChem doi.org/10.1002/cbic.202000100 stress,[10] heavy metal,[11] organic[12] and inorganic compounds,[13] 1 landmine components[14] as well as for drug detection and 2 screening.[15] Application of Lux in reporter genes in eukaryotic 3 systems is challenging because the heterodimeric form of LuxA 4 and LuxB is not suitable for expression in the eukaryotic 5 system.[16] Previously, Vibrio campbellii Lux was engineered to 6 contain a linker which allows the enzyme to be overexpressed 7 in eukaryotic systems.[16c] However, the system still suffers from 8 several drawbacks including the low intensity of the generated 9 light signal and the required use of a flavin reductase to supply 10 the FMNHÀ for the luciferase assays.[16b,c, 17] 11 The auxiliary system for V. campbellii Lux reporter gene 12 assays relies on the use of C reductase which catalyzes the 13 1 reduction of flavin by NADH.[16c,18] The catalytic efficiency of C 14 1 reductase can be enhanced by adding p-hydroxyphenyl acetate Figure 1. The structures of flavin and nicotinamide cofactors. A) Nicotina- 15 mide cofactor structures: natural nicotinamide adenine dinucleotide (NADH) (HPA), an effector of the reductase, to increase the rate of FMN and biomimetic 1-benzyl-1,4-dihydronicotinamide (BNAH). The numbers on 16 À [3a,18] reduction and FMNH transfer. Thus, in addition to the nicotinamide ring illustrate the carbon position. B) Common structures: 17 aldehyde substrate, a reagent cocktail for adding to eukaryotic riboflavin, flavin mononucleotide (FMN), and flavin adenine dinucleotide 18 (FAD). cell lysate needs to contain the C reductase, HPA and NADH to 19 1 produce light emission signals. HPA is a phenolic acid 20 compound with high antioxidant ability and free radical 21 scavenging capacity.[19] It has been reported as a major reaction employing C reductase. Stopped-flow kinetics of FMN, 22 1 metabolite of many natural flavonoids such as proanthocyani- FAD, and riboflavin reduction by BNAH was also investigated to 23 dins and kaempferol regularly found in natural products from explore the fundamental basis of this reaction and measure the 24 fruits and plants.[20] HPA has been reported to rescue or detoxify rate constants of flavin reduction by BNAH. The minimized 25 hepatocellular injury resulting from overdose with acetamino- cascade using BNAH was also used successfully for assaying lux 26 phen (APAP), a common drug for analgesic and antipyretic gene expression in eukaryotic cell lysate, demonstrating that 27 uses. HPA has also been reported to down-regulate key the system can serve as a more simplified and cost-effective 28 enzymes such as cytochrome P450 (CYP) 2E1, which is involved means for conducting bioluminescence assays, which can be 29 in many metabolic processes of xenobiotics. Moreover, HPA can further developed for high-throughput drug and natural 30 stimulate Nrf2 translocation which leads to up-regulation of the product screening platforms. 31 expression of detoxification pathways.[21] Therefore, the extra 32 use of HPA in assay reagents may produce interference with the 33 Lux reactions when used for screening reactive natural 34 Results and Discussion flavonoids compound for investigating novel detoxifiers/inhib- 35 itors/enhancers. We thus explored an alternative method for 36 Stopped-flow experiments to investigate the kinetics of flavin generating FMNH- with fewer enzymatic steps in order to reduction by BNAH 37 circumvent the use of foreign chemicals such as HPA to avoid 38 potential sources of interference, and to reduce assay cost.
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