Chalansonnet Valérie, Mercier Claire, Orenga Sylvain, Gilbert Christophe
bioMérieux, 3 route de port Michaud, 38390, La Balme les Grottes, France.
CIRI, International Center for Infectiology Research, Legionella pathogenesis group, Université de Lyon, Lyon, France.
BMC Microbiol. 2017 May 25;17(1):126. doi: 10.1186/s12866-017-1033-3.
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.
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.
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.
硝基还原酶是依赖NAD(P)H的黄素酶，存在于大多数细菌物种中。尽管粪肠球菌菌株由于对硝基呋喃敏感似乎具有这种活性，但尚未有相关酶被描述。硝基还原酶因其还原硝基化合物的能力而与其他还原酶区分开来。它们根据对辅因子的偏好进一步分类：NADH和/或NADPH。然而，最近，偶氮还原酶因其对硝基化合物，特别是硝基前药的强大活性而受到研究。这一结果表明偶氮和硝基还原酶活性存在交叉。目前，尚未证明有硝基还原酶具有偶氮还原酶活性。但由于与底物相关的序列差异和活性特异性，活性预测并不明显，生化特性鉴定仍然必要。鉴定对这两类化合物（偶氮和硝基）具有活性的酶，有助于考虑它们共同的生理作用。
粪肠球菌V583中的四种假定硝基还原酶EF0404、EF0648、EF0655和EF1181在大肠杆菌中作为带组氨酸标签的重组蛋白过量表达，并按照天然或变性/复性方案进行纯化。EF0648、EF0655和EF1181表现出硝基还原酶活性，它们对辅因子的偏好与其蛋白质序列系统发育一致。EF0404表现出硝基还原酶和偶氮还原酶活性。有趣的是，EF0404的生化特性（底物和辅因子特异性）与已知的偶氮还原酶AzoA的特性相似。但其序列在硝基还原酶组内匹配，与EF0648相同。
我们在此证明了在粪肠球菌V583基因组中鉴定出的假定还原酶的硝基还原酶活性。我们鉴定出了第一种能够直接还原偶氮化合物的硝基还原酶，而其蛋白质序列与其他硝基还原酶接近。因此，这突出了仅根据蛋白质序列比对来对这些酶进行分类的困难，从而强调了通过实验证明活性的必要性。这些结果为考虑这些还原酶更广泛的功能提供了更多数据。
