Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity
Abstract 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 reduct...
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| Format: | Article |
| Language: | English |
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BMC
2017-05-01
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| Series: | BMC Microbiology |
| Subjects: | |
| Online Access: | http://link.springer.com/article/10.1186/s12866-017-1033-3 |
| _version_ | 1811318892372426752 |
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| author | Valérie Chalansonnet Claire Mercier Sylvain Orenga Christophe Gilbert |
| author_facet | Valérie Chalansonnet Claire Mercier Sylvain Orenga Christophe Gilbert |
| author_sort | Valérie Chalansonnet |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-04-13T12:33:09Z |
| format | Article |
| id | doaj.art-f72981bafa8d41b1a06a911731eafe2d |
| institution | Directory Open Access Journal |
| issn | 1471-2180 |
| language | English |
| last_indexed | 2024-04-13T12:33:09Z |
| publishDate | 2017-05-01 |
| publisher | BMC |
| record_format | Article |
| series | BMC Microbiology |
| spelling | doaj.art-f72981bafa8d41b1a06a911731eafe2d2022-12-22T02:46:46ZengBMCBMC Microbiology1471-21802017-05-0117111010.1186/s12866-017-1033-3Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activityValérie Chalansonnet0Claire Mercier1Sylvain Orenga2Christophe Gilbert3bioMérieuxbioMérieuxbioMérieuxCIRI, International Center for Infectiology Research, Legionella pathogenesis group, Université de LyonAbstract 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.http://link.springer.com/article/10.1186/s12866-017-1033-3AzoreductasesNitroreductasesEnterococcus faecalis |
| spellingShingle | Valérie Chalansonnet Claire Mercier Sylvain Orenga Christophe Gilbert Identification of Enterococcus faecalis enzymes with azoreductases and/or nitroreductase activity BMC Microbiology Azoreductases Nitroreductases Enterococcus faecalis |
| 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 | Azoreductases Nitroreductases Enterococcus faecalis |
| url | http://link.springer.com/article/10.1186/s12866-017-1033-3 |
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