Structure and Function of CinD (YtjD) of Lactococcus lactis,a Copper-Induced Nitroreductase Involved in Defense against Oxidative Stress

In Lactococcus lactis IL1403, 14 genes are under the control of the copper-inducible CopR repressor. This so-called CopR regulon encompasses the CopR regulator, two putative CPx-type copper ATPases, a copper chaperone, and 10 additional genes of unknown function. We addressed here the function of one of these genes, ytjD, which we renamed cinD (copper-induced nitroreductase). Copper, cadmium, and silver induced cinD in vivo, as shown by real-time quantitative PCR. A knockout mutant of cinD was more sensitive to oxidative stress exerted by 4-nitroquinoline-N-oxide and copper. Purified CinD is a flavoprotein and reduced 2,6-dichlorophenolindophenol and 4-nitroquinoline-N-oxide with k_cat values of 27 and 11 s⁻¹, respectively, using NADH as a reductant. CinD also exhibited significant catalase activity in vitro. The X-ray structure of CinD was resolved at 1.35 Å and resembles those of other nitroreductases. CinD is thus a nitroreductase which can protect L. lactis against oxidative stress that could be exerted by nitroaromatic compounds and copper.Lactococcus lactis IL1403 is a Gram-positive lactic acid bacterium which is used for the manufacture of food and dairy products but also for an increasing number of biotechnological applications. Given the economical importance of this microorganism, it is often used as a model for molecular studies. Its genome has been sequenced (4), and its proteome has been extensively characterized (11). When applied to industrial processes, this bacterium has to face various stress conditions, such as low pH, high temperature, osmotic shock, and metal stress (44). For instance, in traditional cheese making in Switzerland, L. lactis is exposed to copper released from the copper vats.Copper is an essential micronutrient for both prokaryotes and eukaryotes. The two oxidation states of copper, Cu⁺ and Cu²⁺, allow its participation in many important biological functions. More than 30 enzymes are known to use copper as a cofactor, such as superoxide dismutase (SOD), cytochrome c oxidase, or lysyl oxidase (20). The redox activity of copper can also lead to the generation of free radicals, which cause cellular damage (42, 43). Recently, alternative copper toxicity mechanisms have been demonstrated in bacteria in which copper interferes with the formation of catalytic iron-sulfur clusters (6, 22). Whatever the mechanism of copper toxicity, maintenance of copper homeostasis by controlling the uptake, accumulation, detoxification, and removal of copper is critical for living organisms.Copper homeostasis in L. lactis has not yet been investigated in great detail but appears to resemble the well-characterized copper homeostatic system of Enterococcus hirae (34). L. lactis possesses a copRZA operon, which provides copper resistance. It encodes the CopA copper export ATPase, the CopR copper-inducible repressor, and the CopZ copper chaperone (23). CopR regulates not only the copRZA operon but also an additional 11 genes. This so-called CopR regulon also includes copB, encoding a second putative copper ATPase; lctO, encoding lactate oxidase; and the ydiDE, yahCD-yaiAB, and ytjDBA operons of unknown function. Of all the genes and operons constituting the CopR regulon, the ytjDBA operon was most strongly induced by copper (23). Based on sequence comparison, the first gene of this operon, ytjD, encodes an oxygen-insensitive nitroreductase, which we renamed cinD for copper-induced nitroreductase.Nitroreductases are called oxygen insensitive when they can catalyze the two-electron reduction of nitro compounds in the presence of oxygen. Such enzymes are widespread in nature and are able to reduce a wide range of substrates, such as furazones, nitroaromatic compounds, flavins, and ferricyanide, using NADH or NADPH as the reductant. They are flavoproteins of 22 to 24 kDa and form homodimers with one flavin mononucleotide cofactor per monomer. Although oxygen-insensitive nitroreductases have been extensively studied, their in vivo function remains largely unknown. The closest relative of CinD, which has functionally been studied, is FRP of Vibrio harveyi, with 29% sequence identity to CinD. FRP is not a typical nitroreductase but appears to function as an NADH flavin oxidoreductase which provides reduced flavin to luciferase (19). The next closest relative of CinD, NfsA of Escherichia coli, with 23% sequence identity, exhibits the broad substrate specificity typical of most nitroreductases (48). The structure of this enzyme has been solved at a resolution of 1.7 Å (17). It closely resembles the structures of other enzymes which belong to the oxygen-insensitive nitroreductase family. NfsA has recently been shown to participate in the degradation of 2,4,6-trinitrotoluene (10). This suggests that an important function of nitroreductases could be the metabolism of xenobiotics.We investigated here the structure and function of CinD of L. lactis. CinD was induced by copper, cadmium, and silver and protected L. lactis from oxidative stress exerted by 4-nitroquinoline-N-oxide (NQO). The purified enzyme is a flavoprotein and exhibited nitroreductase activity on NQO and a variety of other substrates, using NADH as the reductant. CinD also possesses catalase activity and is thus able to defend cells against oxidative stress exerted by hydrogen peroxide, xenobiotics, or copper. The three-dimensional structure of CinD was resolved at a 1.35-Å resolution and exhibits a typical nitroreductase structure.