Abstract
BackgroundNitroreductases, 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.ResultsFour 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.ConclusionsWe 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.
Highlights
Nitroreductases, NAD(P)H dependent flavoenzymes, are found in most of bacterial species
Nitroreductase activity in E. faecalis can be hypothesised from the observation that E. faecalis strains are usually sensitive to nitrofurans, antibiotics that are often used in case of urinary tract infections and which have retained value due to the expansion of resistance to ß-lactams [10]
Nitroreductase activity of E. faecalis strains In the combined presence of bacteria and the nitroreductase substrate 7-nitrocoumarin-3-carboxylic acid (7NCCA), an increase of fluorescence was observed (Fig.1)
Summary
Nitroreductases, NAD(P)H dependent flavoenzymes, are found in most of bacterial species. Oxygen-insensitive nitroreductases are a group of flavoenzymes, belonging to oxido-reductases, that are able to reduce nitro compounds depending on nicotinamide adenine dinucleotide availability (NAD(P)H) [1]. In anti-cancer strategy, nitroreductases are one of the most studied candidates for gene-directed enzymeprodrug therapy [9] Due to these potential applications, nitroreductases have been well studied in enteric bacteria, except for Enterococcus faecalis, a Gram positive opportunistic pathogen present in the intestine of a variety of mammals. For this species, nitroreductase activity has never been proven and no nitroreductase enzyme has as yet been characterised. While it appears useful to identify them for potential improvements of such applications
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