Dehydrogenation, oxidative denitration and ring contraction of N,N-dimethyl-5-nitrouracil by a Bacillus nitroreductase Nfr-A1

Nfr-A1, a Bacillus subtilis nitroreductase, catalyzes the nitroreduction of a large panel of aromatic and heterocyclic nitro compounds, except those belonging to nitrouracil class of molecules. Besides nitroreduction, Nfr-A1 exhibits a strong NADH oxidase activity in the presence of oxygen, leading to high concentration of H2O2 (up to 200 μM). In the presence of (N,N)-dimethyl-5-nitrouracil 1 (dim-NU), Nfr-A1 achieves the reduction of dim-NU double bond to compounds 2 and 3 and in parallel the oxidation of dim-NU to the denitrated five membered derivatives 4 and 5. The reduction is catalyzed by the reduced flavin Fl-Red and resembles those catalyzed by dihydropyrimidine dehydrogenases (DPD), during the catabolism of pyrimidines. The oxidative denitration is catalyzed in part by hydrogen peroxide generated through the NADH-oxidase activity, and certainly by the peroxyflavin intermediate Fl-OOH for the other part. The mechanisms of reaction were proposed according to experimental data and literature. These findings together with our previous results on the potential biological role of Nfr-A1, confirm the large spectrum of catalysis supported by this enzyme. The oxidative denitration is sporadically reported in literature and represents a safe and green alternative for the remediation of nitro-compounds.

NfrA1, the oxygen insensitive Bacillus subtilis nitroreductase, catalyzes in the presence of NADH, the reduction and oxidation of (N,N)-dimethyl-5-nitrouracil 1 (DimNU). The reduction affects the double bound leading to compounds 2 and 3. The oxidation involves a concomitant ring contraction and denitration to 4 and 5. The reactive forms are the oxidized and reduced flavin and the mechanism that accounts for the formation of metabolites is proposed.Figure optionsDownload as PowerPoint slideHighlights
► Nfr-A1 is a nitroreductase from B. subtilis with a large catalytic spectrum.
► Nfr-A1 reduces the double bound of (N,N)-dimethyl-uracil (dimNU).
► Nfr-A1 catalyzes the oxidative denitration of dimNU followed by ring contraction.
► The reduction is supported by the reduced flavin intermediate.
► The oxidation is catalyzed by both the hydroperoxyflavin and hydrogen peroxide.