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Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity

BACKGROUND: Nitroreductases, NAD(P)H dependent flavoenzymes, are found in most of bacterial species. Even if Enterococcus faecalis strains seems to present such activity because of their sensitivity to nitrofurans, no enzyme has been described. Nitroreductases were separated of others reductases due...

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Detalles Bibliográficos
Autores principales: Chalansonnet, Valérie, Mercier, Claire, Orenga, Sylvain, Gilbert, Christophe
Formato: Online Artículo Texto
Lenguaje:English
Publicado: BioMed Central 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445473/
https://www.ncbi.nlm.nih.gov/pubmed/28545445
http://dx.doi.org/10.1186/s12866-017-1033-3
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author Chalansonnet, Valérie
Mercier, Claire
Orenga, Sylvain
Gilbert, Christophe
author_facet Chalansonnet, Valérie
Mercier, Claire
Orenga, Sylvain
Gilbert, Christophe
author_sort Chalansonnet, Valérie
collection PubMed
description BACKGROUND: Nitroreductases, NAD(P)H dependent flavoenzymes, are found in most of bacterial species. Even if Enterococcus faecalis strains seems to present such activity because of their sensitivity to nitrofurans, no enzyme has been described. Nitroreductases were separated of others reductases due to their capacity to reduce nitro compounds. They are further classified based on their preference in cofactor: NADH and/or NADPH. However, recently, azoreductases have been studied for their strong activity on nitro compounds, especially nitro pro-drugs. This result suggests a crossing in azo and nitro reductase activities. For the moment, no nitroreductase was demonstrated to possess azoreductase activity. But due to sequence divergence and activity specificity linked to substrates, activity prediction is not evident and biochemical characterisation remains necessary. Identifying enzymes active on these two classes of compounds: azo and nitro is of interest to consider a common physiological role. RESULTS: Four putative nitroreductases, EF0404, EF0648, EF0655 and EF1181 from Enterococcus faecalis V583 were overexpressed as his-tagged recombinant proteins in Escherichia coli and purified following a native or a denaturing/renaturing protocol. EF0648, EF0655 and EF1181 showed nitroreductase activity and their cofactor preferences were in agreement with their protein sequence phylogeny. EF0404 showed both nitroreductase and azoreductase activity. Interestingly, the biochemical characteristics (substrate and cofactor specificity) of EF0404 resembled the properties of the known azoreductase AzoA. But its sequence matched within nitroreductase group, the same as EF0648. CONCLUSIONS: We here demonstrate nitroreductase activity of the putative reductases identified in the Enterococcus faecalis V583 genome. We identified the first nitroreductase able to reduce directly an azo compound, while its protein sequence is close to others nitroreductases. Consequently, it highlights the difficulty in classifying these enzymes solely on the basis of protein sequence alignment and hereby the necessity to experimentally demonstrate the activity. The results provide additional data to consider a broader functionality of these reductases.
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spelling pubmed-54454732017-05-30 Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity Chalansonnet, Valérie Mercier, Claire Orenga, Sylvain Gilbert, Christophe BMC Microbiol Research Article BACKGROUND: Nitroreductases, NAD(P)H dependent flavoenzymes, are found in most of bacterial species. Even if Enterococcus faecalis strains seems to present such activity because of their sensitivity to nitrofurans, no enzyme has been described. Nitroreductases were separated of others reductases due to their capacity to reduce nitro compounds. They are further classified based on their preference in cofactor: NADH and/or NADPH. However, recently, azoreductases have been studied for their strong activity on nitro compounds, especially nitro pro-drugs. This result suggests a crossing in azo and nitro reductase activities. For the moment, no nitroreductase was demonstrated to possess azoreductase activity. But due to sequence divergence and activity specificity linked to substrates, activity prediction is not evident and biochemical characterisation remains necessary. Identifying enzymes active on these two classes of compounds: azo and nitro is of interest to consider a common physiological role. RESULTS: Four putative nitroreductases, EF0404, EF0648, EF0655 and EF1181 from Enterococcus faecalis V583 were overexpressed as his-tagged recombinant proteins in Escherichia coli and purified following a native or a denaturing/renaturing protocol. EF0648, EF0655 and EF1181 showed nitroreductase activity and their cofactor preferences were in agreement with their protein sequence phylogeny. EF0404 showed both nitroreductase and azoreductase activity. Interestingly, the biochemical characteristics (substrate and cofactor specificity) of EF0404 resembled the properties of the known azoreductase AzoA. But its sequence matched within nitroreductase group, the same as EF0648. CONCLUSIONS: We here demonstrate nitroreductase activity of the putative reductases identified in the Enterococcus faecalis V583 genome. We identified the first nitroreductase able to reduce directly an azo compound, while its protein sequence is close to others nitroreductases. Consequently, it highlights the difficulty in classifying these enzymes solely on the basis of protein sequence alignment and hereby the necessity to experimentally demonstrate the activity. The results provide additional data to consider a broader functionality of these reductases. BioMed Central 2017-05-25 /pmc/articles/PMC5445473/ /pubmed/28545445 http://dx.doi.org/10.1186/s12866-017-1033-3 Text en © The Author(s). 2017 Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.
spellingShingle Research Article
Chalansonnet, Valérie
Mercier, Claire
Orenga, Sylvain
Gilbert, Christophe
Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity
title Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity
title_full Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity
title_fullStr Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity
title_full_unstemmed Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity
title_short Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity
title_sort identification of enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445473/
https://www.ncbi.nlm.nih.gov/pubmed/28545445
http://dx.doi.org/10.1186/s12866-017-1033-3
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