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Reduction of polynitroaromatic compounds: the bacterial nitroreductases

María Dolores Roldán, Eva Pérez-Reinado, Francisco Castillo, Conrado Moreno-Vivián
DOI: http://dx.doi.org/10.1111/j.1574-6976.2008.00107.x 474-500 First published online: 1 May 2008


Most nitroaromatic compounds are toxic and mutagenic for living organisms, but some microorganisms have developed oxidative or reductive pathways to degrade or transform these compounds. Reductive pathways are based either on the reduction of the aromatic ring by hydride additions or on the reduction of the nitro groups to hydroxylamino and/or amino derivatives. Bacterial nitroreductases are flavoenzymes that catalyze the NAD(P)H-dependent reduction of the nitro groups on nitroaromatic and nitroheterocyclic compounds. Nitroreductases have raised a great interest due to their potential applications in bioremediation, biocatalysis, and biomedicine, especially in prodrug activation for chemotherapeutic cancer treatments. Different bacterial nitroreductases have been purified and their biochemical and kinetic parameters have been determined. The crystal structure of some nitroreductases have also been solved. However, the physiological role(s) of these enzymes remains unclear. Nitroreductase genes are widely spread within bacterial genomes, but are also found in archaea and some eukaryotic species. Although studies on regulation of nitroreductase gene expression are scarce, it seems that nitroreductase genes may be controlled by the MarRA and SoxRS regulatory systems that are involved in responses to several antibiotics and environmental chemical hazards and to specific oxidative stress conditions. This review covers the microbial distribution, types, biochemical properties, structure and regulation of the bacterial nitroreductases. The possible physiological functions and the biotechnological applications of these enzymes are also discussed.

  • bacteria
  • cancer therapy
  • flavoproteins
  • nitroaromatics
  • nitroreductases
  • SoxRS/MarRA regulatory systems


Toxic compounds can be classified into natural products, which are generated as a consequence of the metabolic activities of living organisms, and xenobiotic chemicals, which are mainly produced by industrial processes or other human activities (Hartter, 1985; Spain, 1995; Peres & Agathos, 2000; Rieger et al., 2002). Most xenobiotic compounds are recalcitrant and persist because they contain structures or substituents that are not normally present in natural compounds and limit their biodegradability. Owing to their human origin, xenobiotic compounds have been released to the environment very recently, and therefore, only a small number of organisms in nature have developed the capability to degrade these molecules (Crawford, 1993, 1995; Spain et al., 1995; Peres & Agathos, 2000; Esteve-Núñez, 2001; Heiss & Knackmuss, 2002; Rieger et al., 2002; Ramos et al., 2005).

Only a few nitroaromatic compounds are natural products (Fig. 1), such as chloramphenicol (Ahmed & Vining, 1983), nitropyoluteorin (Ohmori et al., 1978), oxypyrrolnitrin (Hashimoto & Hattori, 1966; Kirner et al., 1998) and phidolopin (Tischler et al., 1986). Some plants like Astragalus also synthesize nitroglycosides as defense mechanism (Anderson et al., 1993). Most nitroaromatic compounds are generated as a consequence of industrial processes focused on the synthesis of polyurethanes, pesticides like parathion (diethyl-p-nitrophenylmonothiophosphate) and dinoseb (2-sec-butyl-4,6-dinitrophenol), and explosives such as 2,4,6-trinitrotoluene (TNT) and picric acid or 2,4,6-trinitrophenol (Fig. 1). Mono- and di-nitrophenols are also generated in the synthesis of plastics, dyes, solvents and drugs (Hartter, 1985; Spain, 1995; Peres & Agathos, 2000).

Figure 1

Chemical structures of relevant nitro-compounds including natural (top) and nonnatural or xenobiotic (bottom) nitroaromatic, nitroester and nitroheterocyclic compounds.

Nitroaromatic compounds are released to the environment during manufacturing and handling, through filtration and losses of the storage tanks and during transport and intensive military activities. Presently there are vast areas worldwide polluted with these compounds, especially with TNT and other explosives (Fig. 1; Hawari et al., 2000a; Rieger et al., 2002; Lewis et al., 2004; Ramos et al., 2005). Polynitroaromatic compounds can be adsorbed and retained in the soils by clay and humic materials (Haderlein & Schwarzenbach, 1993), although biotransformation of TNT produces amino derivatives that are much more strongly adsorbed in soils than TNT (Daun et al., 1998; Lenke et al., 1998; Ahmad & Hughes, 2002; Rieger et al., 2002; Lewis et al., 2004). The immobilized nitroaromatics and derivatives exhibit decreased bioavailability and toxicity. However, some of these strongly adsorbed compounds may be accessible for microbial metabolism if the conditions for an adequate population of microorganisms with degradative capabilities are optimized (Robertson & Jemba, 2005). Nitroaromatic compounds are also found in groundwater, as a consequence of leaching of contaminated soils, and in the atmosphere, due to combustion processes (Mori et al., 2003). Aromatic hydrocarbons or nitroarenes may be formed in the atmosphere by photochemical transformation of polyaromatic hydrocarbons (Crawford, 1993).

Mononitroaromatic compounds can be degraded by many different bacterial strains, but polynitrated aromatic compounds are recalcitrant chemicals difficult to degrade. In addition, microorganisms able to degrade polysubstituted nitroaromatic compounds grow at a very slow rate, probably due to the high toxicity and low solubility of these chemicals (Ramos et al., 2005). Nitroreductases are NAD(P)H-dependent flavoenzymes that catalyze the reduction of the nitro groups of a great variety of polynitrated aromatic compounds. Genes coding for nitroreductase-like proteins are present in most bacterial genomes, and are also found in archaea and eukaryotic organisms (Marques de Oliveira, 2007). Bacterial nitroreductases have received a lot of attention in the last few decades due to their potential environmental and clinical biotechnological applications. Bioremediation treatments for polynitrated aromatic compounds, including composting and bioslurry processes, and phytoremediation using transgenic plants bearing bacterial nitroreductases, may be attractive and effective decontaminating procedures that can be performed in situ (Spain, 1995; Lenke et al., 1998; French et al., 1999; Hawari et al., 2000a, b; Hannink, 2001, 2002; Rieger et al., 2002; Schrader & Hess, 2004; Ramos et al., 2005). In addition, bacterial nitroreductases have a great clinical interest because they are used for cancer therapy in the techniques known as antibody-directed enzyme pro-drug therapy (ADEPT), gene-directed enzyme pro-drug therapy (GDEPT) or virus-directed enzyme pro-drug therapy (VDEPT). These treatments are based on the use of an Escherichia coli nitroreductase, or its gene, to sensitize the tumor cells to a prodrug that is converted by the bacterial enzyme into highly cytotoxic hydroxylamine derivatives that cause tumor cell killing (Knox et al., 1993; Knox & Connors, 1995, 1997; Green et al., 2004; Searle et al., 2004). In this review are presented and discussed the most relevant aspects of the reduction of polynitroaromatic compounds, with special emphasis on the structure, biochemical mechanisms, physiological functions and regulatory properties of the bacterial nitroreductases, and their potential biotechnological applications.

Toxicity of nitroaromatics

Many nitroaromatic compounds (Fig. 1) are toxic and mutagenic, as revealed by different studies on several organisms, including bacteria, algae, plants, invertebrates and mammals (Won et al., 1976; Whong & Edwards, 1984; Honeycutt et al., 1996; Lachance et al., 1999; Robidoux et al., 1999; Schäfer & Achazi, 1999; Frische et al., 2002, 2003). The toxicity of nitro-compounds is associated with the products formed during reduction of the nitro groups. The hydroxylamino derivatives can interact with biomolecules, including DNA, causing toxic and mutagenic effects. The toxic effects are related to the electrophilic character of these derivatives, whereas the mutagenic effects are mainly due to the formation of hydroxylamine moiety adducts through esterification with guanine (Corbett & Corbett, 1995). It has been proposed that genotoxic effects of arylamines are associated with their conversion to hydroxylamino derivatives (Cramer et al., 1960). In mammals, nitroaromatic compounds are transformed into conjugated metabolites that are further dissociated and/or reduced by the intestinal microbial communities. The nitroreductases present in the intestinal microbial communities play a key role in the metabolism of exogenous nitroaromatic chemicals to which the host is exposed. Studies on axenic and nonaxenic animals have revealed the involvement of nitroreductases in the transformation of these chemicals into carcinogenic metabolites due to reduction of the nitro groups to the corresponding hydroxylamino derivatives (Blumer et al., 1980). The mutagenic effects are caused by the production of arylhydroxylamines or acetoxyarylamines and the electrophilic nitrenium ion (−N+), which can interact with DNA (Fu, 1990). Some chemical carcinogens like N-nitroso compounds need to be initially metabolized to induce mutagenicity. The nitroreductases from the intestinal microbial communities stimulate toxicity while acetyltransferases stimulate mutagenicity of these compounds (Aiub et al., 2006). 1-Nitropyrene (Fig. 1) is transformed through hydroxylation of the aromatic ring and further reduction by the intestinal microbial communities, although the N-acetylation of the amino derivatives occurs basically in the liver (Kinouchi & Ohnishi, 1983). In the case of dinitropyrenes, adducts with DNA are only produced when the nitro group is reduced, because oxygenation of the aromatic ring is less favorable in the presence of two nitro groups. The toxic effects can be produced as a result of the reaction of arylamines with other molecules, such as oxyhemoglobin. Both, arylamine and oxyhemoglobin are oxidized in the presence of oxygen, forming a redox cycling which produces ferrihemoglobin and hydroxylamine, which can react again with oxyhemoglobin (Maples et al., 1990). Nitrosoarenes may react with the sulfhydryl groups, and cause protein inactivation (Eyer et al., 1979). Binding of TNT to proteins also causes cytotoxic effects in the liver (Liu et al., 1992). Formation of hemoglobin adducts of TNT amino-derivatives has also been found in humans exposed to this explosive, and genotoxicity and potential carcinogenicity of TNT have been reported (Bolt et al., 2006). In addition, some nitroaromatic compounds are toxic per se and act as uncouplers of oxidative phosphorylation, inhibiting the generation of the proton motive force required for ATP synthesis, as occurs in the case of 2,4-dinitrophenol and picric acid (Fig. 1; Hanstein & Hatefi, 1974).

Reductive degradation pathways of nitroaromatic compounds

The chemical stability of nitroaromatic compounds is based on the delocalization of the electrons in the aromatic ring. Despite this stability, microorganisms have found the way of breaking-down the aromatic ring to produce intermediates of central metabolic pathways. Oxygen is the reactive molecule that promotes the breakdown of many aromatic compounds, which are metabolized by reactions catalyzed by mono- and di-oxygenases (Spain, 1995). However, the pronounced electron-withdrawing character of the nitro groups in the polynitroaromatic compounds causes an electron deficiency in the aromatic ring that favors initial reductive reactions. Oxidative attack of nitroaromatic compounds takes place under aerobic conditions, whereas reductive metabolism of polynitroaromatic compounds may occur both aerobically and anaerobically. Thus, hydroxyl derivatives formation by oxidation of the aromatic ring via mono- and di-oxygenases, followed by aromatic ring opening, is the expected mechanism for aerobic degradation of mononitroaromatic compounds, and even for some dinitroaromatics (Spanggord et al., 1991; Haigler & Spain, 1993; Nishino & Spain, 1993; Suen & Spain, 1993; Haigler et al., 1994; Suen et al., 1996; Roldán, 1998; Spiess et al., 1998; Zablotowicz et al., 1999; Nishino et al., 2000). The possibility of oxidative degradation decreases if the number of nitro substituents increases, because nitro groups withdraw electrons from the aromatic ring, leaving it electron-deficient and impairing the electrophilic attack mediated by oxygenases. Consequently, polynitrated aromatic compounds like TNT and picric acid, an explosive structurally similar to TNT (Fig. 1), are usually degraded by reductive pathways. The reduction rate of a nitroaromatic compound is also determined by the chemical properties of other substituents present in the aromatic ring. Thus, the rate of the nitro group reduction increases when other group is present in para position, with the following priority NH2<OH<H<CH3<COOH<NO2 (McCormick et al., 1976). Therefore, reduction of the first nitro group to amino group implies a difficulty for other nitro substituents to be further reduced. Aminodinitrotoluenes (ADNT) and diaminonitrotoluenes (DANT) have been described as the main intermediates in the biotransformation of TNT, but the complete reduction of TNT to triaminotoluene (TAT) has been described only under strict anaerobic conditions (Lewis et al., 1996; Hawari et al., 2000a; Esteve-Núñez, 2001; Rieger et al., 2002). A large number of microorganisms able to transform TNT and other polynitrated aromatic compounds, either aerobically or anaerobically, have been described (reviewed in Esteve-Núñez, 2001). Bacterial reduction of the nitro groups of polynitrated aromatic compounds may be a gratuitous cometabolic process that requires an exogenous carbon source to provide reducing equivalents (Peres & Agathos, 2000; Rieger et al., 2002). However, in many cases, nitroaromatic compounds are used as a source of nitrogen for bacterial growth (Boopathy & Kulpa, 1992; Johnson & Spain, 2003; Caballero et al., 2005a; Caballero & Ramos, 2006). Also, it has been reported that TNT reduction in the strain Pseudomonas putida JLR11 is coupled to the generation of an electrochemical proton gradient that is sufficient to drive ATP synthesis (Esteve-Núñez & Ramos, 1998; Esteve-Núñez, 2000).

Polynitroaromatic compounds may be transformed through two types of reductive pathways: (1) the reduction of the aromatic ring by addition of hydride ions through hydride transferases to generate the so-called hydride–Meisenheimer complexes, which may be further metabolized with the concomitant release of nitrite, or (2) the reduction of the nitro groups to hydroxylamino or amino groups, catalyzed by nitroreductases (Fig. 2). The amino compounds could also be formed as end products from the hydroxylamino derivatives by a Bamberger-type rearrangement (Spain, 1995). Nitrite or ammonium released from the aromatic ring by futher degradative pathways can be used as nitrogen source for bacterial growth (Fig. 2).

Figure 2

Pathways for reduction of polynitroaromatic compounds in bacteria. There are two main pathways for reduction of polynitrated aromatic compounds: the reduction of the aromatic ring through addition of two hydride ions to generate the dihydride–Mesenheimer complex, which is catalyzed by hydride transferases or by some enzymes of the OYE family, and the reduction of the nitro groups through successive addition of electrons, catalyzed by different enzymes with nitroreductase activity. In both cases an inorganic nitrogen source, nitrite or ammonium, may be released and used for bacterial growth. R, –CH3 (TNT) or –OH (picric acid).

Some Rhodococcus and Nocardioides strains can mineralize 2,4-dinitrophenol and picric acid by a degradative pathway that involves two hydride transferases (HTII and HTI) and an NADPH-dependent F420 reductase (Lenke & Knackmuss, 1992, 1996; Lenke et al., 1992; Vorbeck et al., 1994, 1998; Ebert et al., 1999, 2001; Rieger et al., 1999). These proteins show high similarity to the corresponding enzymes of methanogenic archaea (Ebert et al., 2001; Heiss et al., 2002a, b). Hydride transferase II (HTII, encoded by the npdI gene) is required for the first hydride transfer to the aromatic ring that produces a hydride–Mesenheimer complex, and hydride transferase I (HTI, encoded by the npdC gene) catalyzes a second hydride transfer that originates the dihydride–Meisenheimer complex (Vorbeck et al., 1994, 1998; Hoffmann et al., 2004). The activity of both transferases requires the NADPH-dependent F420 reductase (NDFR, encoded by the npdG gene) to supply the hydride ions for the synthesis of both hydride and dihydride–Meisenheimer complexes (Ebert et al., 2001). In Nocardioides simplex, a single hydride transferase catalyzes both hydrogenation reactions (Ebert et al., 2001). To complete the degradative pathway, a tautomerase (encoded by the npdH gene) interconverts the aci-nitro and the nitro forms of the dihydride–Meisenheimer complex, and nitrite is further removed by a denitrase activity, which only eliminates nitrite from the aci-nitro form of the complex (Hoffmann et al., 2004). Finally, a hydrolase (the npdF gene product) is also required to convert 2,4-dinitrocyclohexanone into 4,6-dinitrohexanoate before the complete mineralization of the nitroaromatic compound (Ebert et al., 2001; Heiss et al., 2002a, b; Hoffmann et al., 2004). In Rhodococcus sp. RB1, mineralization of 2,4-dinitrophenol generates 3-nitroadipate as an intermediate, but this compound may be produced by removal of the nitro group either from the dihydride–Meisenheimer complex or from 4,6-dinitrohexanoate (Blasco et al., 1999). The Rhodococcus opacus npd gene cluster includes a gene (npdR) coding for a transcription regulator of the IclR family (Molina-Henares et al., 2006) that represses npd gene expression. The NpdR repressor becomes inactivated by the binding of different nitroarenes, and this allows the expression of the npd genes required for the degradative pathway (Nga et al., 2004).

On the other hand, reduction of the nitro groups is catalyzed by NAD(P)H-dependent bacterial nitroreductases with broad substrate specificity. These enzymes contain flavin mononucleotide (FMN) as a prosthetic group and usually generate the hydroxylamino or the amino derivative as the end product. According to their sensitivity to oxygen, bacterial nitroreductases may be classified into type I or oxygen-insensitive and type II or oxygen-sensitive enzymes. The biochemical basis that determines whether a nitroreductase is oxygen sensitive or insensitive, and whether it produces hydroxylamino or amino derivatives as the end product, are discussed below. Some other bacterial enzymes also show nitroreductase activity. Thus, in the strictly anaerobic bacterium Clostridium acetobutylicum a Fe-hydrogenase catalyzes the H2-dependent reduction of TNT to hydroxylamino intermediates during the acidogenic growth phase. This Fe-only hydrogenase has been purified and shows an apparent Km value for TNT of 152 μM (Watrous et al., 2003). Also, TNT reduction by carbon monoxide dehydrogenase has been reported in Clostridium thermoaceticum (Huang et al., 2000). However, these clostridial enzymes with nitroreductase activity are structurally and biochemically different from the classical NAD(P)H-dependent nitroreductases that are also present in these bacteria.

Some flavoreductases of the so-called old yellow enzyme (OYE) family use a broad range of substrates including quinones, cytochrome c, methylene blue, ferricyanide, and Fe3+. Also, the OYE enzymes are capable of denitrating some explosives such as the nitroesters pentaerythritol tetranitrate (PETN) and glycerol trinitrate (GTN) and the polynitroaromatic TNT (Fig. 1). The physiological reductant of OYE is assumed to be NADPH. The PETN reductase of Enterobacter cloacae, the NemA protein of Escherichia coli, the xenobiotic reductases XenA and XenB of P. putida and Pseudomonas fluorescens, and the morphinone reductase of P. putida are well known members of this OYE family (Pak et al., 2000; Williams & Bruce, 2002; Williams et al., 2004; Ramos et al., 2005). The OYE proteins may attack polynitroaromatic compounds, such as TNT, by reducing either the aromatic ring to generate hydride– and dihydride–Meisenheimer complexes with the subsequent release of nitrite, or the nitro groups to produce the hydroxylamino derivatives. All bacterial OYE enzymes seem to be able to reduce the nitro groups of polynitroaromatic compounds, but only some of them, like the P. putida xenobiotic reductase XenB, the Escherichia coli NemA protein, and the Enterobacter cloacae PETN reductase, reduce both the nitro groups and the aromatic ring (Williams & Bruce, 2002; Williams et al., 2004; Ramos et al., 2005). In addition, some members of the OYE family are capable to degrade nonaromatic compounds, like the nitroesters PETN or GTN, by a mechanism which includes a reductive denitration with the release of nitrite (Williams & Bruce, 2002). The crystal structures of different OYE enzymes have been solved (Fox & Karplus, 1994; Barna et al., 2001; Breithaupt et al., 2001; Khan et al., 2002, 2004; Orville et al., 2004a, b), but their physiological roles remain unclear. These OYE flavoproteins will not be further considered because they do not show significant amino acid sequence similarity to the typical bacterial nitroreductases and have been recently reviewed (Williams & Bruce, 2002; Williams et al., 2004).

Bacterial nitroreductases

Types of bacterial nitroreductases

The reduction of the nitro groups of polynitroaromatic compounds can be performed through one- or two-electron mechanism. Therefore, two types of bacterial nitroreductases have been described, which also differ in their response to oxygen (Fig. 3). The bacterial oxygen-insensitive or type I nitroreductases catalyze the sequential reduction of the nitro groups through the addition of electron pairs from NAD(P)H to produce the nitroso, hydroxylamino and amino derivatives (Peterson et al., 1979; Bryant et al., 1981). However, the hydroxylamino derivative may be the end product of the reaction and usually, the nitroso intermediate is not detected because the second two-electron reaction has a much faster rate than the first two-electron transfer (Koder et al., 2002; Race et al., 2005). In mammals, the NAD(P)H:quinone reductase or DT-diaphorase (Knox et al., 1993) and the xanthine oxidase (Ueda et al., 2003) also catalyze this type of reaction. On the other hand, the oxygen-sensitive or type II nitroreductases catalyze the single-electron reduction of the nitro group to produce a nitro anion radical, which can be reoxidized aerobically to the original structure with the concomitant production of the superoxide anion in a futile cycle (Fig. 3). Oxygen-sensitive nitroreductases are found in Escherichia coli (Mason & Holtzman, 1975; Peterson et al., 1979) and several Clostridium strains (Angermaier & Simon, 1983). In addition, some enzymes like NADPH:cytochrome P450 reductase (Orna & Mason, 1989), ferredoxin:NADP+ reductase (Anuseviĉius, 1997) and NADH:ubiquinone reductase (Bironaitè, 1991) also catalyze this type of reduction. The mammalian selenoprotein thioredoxin reductase also has nitroreductase activity either with one- or two-electron chemistry, and the single-electron reaction leads to superoxide anion formation (Ĉènas, 2006). Bacteria may contain both types of nitroreductases, although the best-studied enzymes belong to type I because a great variety of them have been purified and/or their genes cloned and characterized. Therefore, the following information presented in this review is focused on the bacterial oxygen-insensitive or type I nitroreductases.

Figure 3

Oxygen-sensitive and -insensitive nitroreductases. The oxygen-sensitive nitroreductases (type II) catalyze the reduction of the nitro group of nitroaromatic compounds by the addition of one electron, forming a nitro anion radical, which in the presence of oxygen generates the superoxide anion in a futile cycle that regenerates the nitro group. The oxygen-insensitive nitroreductases (type I) catalyze the reduction of the nitro group of nitroaromatics by addition of pair of electrons forming the nitroso and the hydroxylamino intermediates before the formation of the amino group. The nitroso intermediate is not usually observed and, in many cases, the hydroxylamino derivative is the end product of the enzymatic reaction. The aromatic ring may contain several substituents. To simplify, only one nitro group is shown.

The biochemical characterization of the type I nitroreductases from enterobacteria can be taken as a model to understand their oxygen insensitivity and the facts that determine the production of the hydroxylamino derivative rather than the amino derivative as the end product. Oxygen-insensitive nitroreductases are very specific for two-electron transfers due to flavin thermodynamics in the enzyme cofactor, so that the one-electron-reduced semiquinone state of both the free-enzyme and the enzyme-substrate complex are strongly suppressed (Koder et al., 2002). Type I nitroreductases preferentially perform two-electron chemistry with a ping–pong bi–bi kinetic mechanism, and the FMN group cycles between the oxidized and the reduced states with a flavin two-electron reduction midpoint potential near that of free FMN (about −190 mV in the Enterobacter cloacae NR nitroreductase). Therefore, the single-electron reaction and the formation of semiquinone are not stabilized and the enzyme does not transfer one electron to oxygen to generate the superoxide anion, as occurs in the type II nitroreductases (Koder et al., 2002). Four electron transfer to the substrate nitrobenzene by the NR enzyme of Enterobacter cloacae produces hydroxylaminobenzene (Fig. 1), which could be further reduced to aniline in a two-electron reaction at a potential highly negative. However, the end product of nitrobenzene transformation is the hydroxylamino derivative because the NR enzyme midpoint potential is not low enough to reduce hydroxylaminobenzene to aniline (Koder et al., 2002).

Bacterial distribution and biochemical properties of oxygen-insensitive nitroreductases

Nitroreductases are widely distributed among bacteria, but nitroreductase-like proteins are also found in archaea and eukaryotic organisms. In addition, most bacteria contain several types of nitroreductases. Thus, four different nitroreductases have been isolated from the gut microbial community organism Bacteroides fragilis (Kinouchi & Ohnishi, 1983). Nitroreductases were first described in bacteria able to reduce chloramphenicol and p-nitrobenzoic acid to their respective amines (Saz & Martínez, 1956; Cartwright & Cain, 1959; Villanueva et al., 1964), but the relevance acquired by these enzymes in the last few decades has lead to the identification and characterization of different nitroreductases from many microorganisms, including gram-negative and gram-positive bacteria; symbionts, pathogens and free-living organisms; heterotrophs and phototrophs; mesophilic and thermophilic species; and aerobic, anaerobic and facultative bacteria. The most relevant bacterial nitroreductases are listed in Table 1, and among them, the enzymes from enteric bacteria are especially well studied. In addition, many putative bacterial nitroreductase genes have been identified and sequenced. The phylogenetic analysis of these sequences suggests that type I or oxygen-insensitive nitroreductases can be classified into two main groups or families that are represented by the Escherichia coli nitroreductases NfsA (group A) and NfsB (group B), respectively (Fig. 4). Group A nitroreductases are usually NADPH-dependent, whereas group B nitroreductases may use both NADH and NADPH as electron donors. Almost all nitroreductases share similar biochemical properties. They usually occur as homodimers (24–30 kDa subunits), have broad substrate specificity, contain FMN as cofactor and catalyze the reduction of the different nitrocompounds using a ping–pong bi–bi kinetic mechanism. Nitroreductases are strongly inhibited by dicoumarol, a diaphorase activity inhibitor, and by p-hydroxymercuribenzoate, a sulphydryl group reagent. Other specific inhibitors are p-iodosobenzoic acid, sodium azide and Cu2+ ions.

View this table:
Table 1

Relevant bacterial oxygen-insensitive nitroreductases

Bacteria and nitroreductase namesMonomer sizes (kDa)Main substratesElectron donorsReferences
Escherichia coli NfsA27Nitrofurazone and other nitro-compounds, chromateNADPHBryant et al. (1981), Zenno (1996a,1998a)
Escherichia coli NfsB24Nitrofurazone and different nitro-compounds (including CB1954 and CL-20), chromateNAD(P)HBryant et al. (1981), Zenno (1996b, 1998c)
Samonella enterica Cnr24Different nitroaromatics (p-nitrophenol, p-nitrobenzoate, 1-nitropyrene), 2-aminofluorene, menadione, flavinsNAD(P)HWatanabe (1990, 1998)
Salmonella enterica SnrA28Different nitroaromatic and nitroheterocyclic compoundsNADPHNokhbeh et al. (2002)
Enterobacter cloacae NR27Nitrofurans, nitrobenzenes, quinones, nitroimidazoles, TNTNAD(P)HBryant & DeLuca (1991), Koder & Miller (1998)
Klebsiella sp. NTR I27TNT, 2,4-dinitrotolueneNAD(P)HKim et al. (2003), Kim & Song (2005)
Pseudomonas pseudoalcaligenes NbzA30Nitrobenzene, TNT, 4-nitrobiphenyl etherNADPHSomerville et al. (1995), Fiorella & Spain (1997), Nadeau & Spain (2000)
Pseudomonas putida PnrA28TNT, 2,4-dinitrotoluene, 4-nitrotoluene, 4-nitrobenzoate, 3,5-dinitroanilineNADPHCaballero et al. (2005b)
Vibrio fischeri FRaseI25FMN, quinones, different nitro-compoundsNAD(P)HZenno et al. (1994)
Vibrio harveyi FRP26FMN, different nitro-compounds, chromateNADPHLei et al. (1994)
Synechocystis sp. DrgA26Flavins, quinones, ferric ion, nitrofurazone, dinosebNAD(P)HMatsuo et al. (1998), Takeda et al. (2007)
Rhodobacter capsulatus NprA272,4-dinitrophenol and several nitroaromatic and nitroheterocyclic compoundsNAD(P)HBlasco & Castillo (1993), Pérez-Reinado (2005)
Bacillus subtilis YwrO22CB1954NAD(P)HAnlezark et al. (2002)
Bacillus subtilis NfrA1(YwcG)29Flavins, nitrofurazone, nitrofurantoinNADPHZenno et al. (1998b)
Staphylococcus aureus NfrA29Flavins, nitrofurazone, nitrofurantoinNADPHStreker et al. (2005)
Clostridium acetobutylicum NitA31TNT, 2,4-dinitrotolueneNADHKutty & Bennett (2005)
Clostridium acetobutylicum NitB23TNT, 2,4-dinitrotolueneNAD(P)HKutty & Bennett (2005)
Figure 4

Phylogenetic tree of the oxygen-insensitive or type I nitroreductases. There are two main groups or families of oxygen-insensitive nitroreductases. The group A includes the major nitroreductase NfsA of Escherichia coli (Zenno et al., 1996a) and the homologous proteins SnrA of Salmonella typhimurium (Nokhbeh et al., 2002), PnrA of Pseudomonas putida JLR11 (Caballero et al., 2005b) and FRP of Vibrio harveyi, an NADPH-flavin oxidoreductase (Lei et al., 1994). The group B includes two subgroups the B1 subfamily with the Escherichia coli minor nitroreductase NfsB (Zenno et al., 1996b), the NR and RNR proteins of Enterobacter cloacae (Bryant et al., 1991; Koder et al., 2001), the classical nitroreductase Cnr of Salmonella typhimurium (Watanabe et al., 1990), the PnrB nitroreductase from P. putida JLR11 (Caballero et al., 2005b), the NAD(P)H-flavin oxidoreductase FRaseI of Vibrio fischeri (Zenno et al., 1994), the Synechocystis DrgA quinone reductase (Matsuo et al., 1998; Takeda et al., 2007), and the major nitroreductase NprA of the phototrophic bacterium Rhodobacter capsulatus (Pérez-Reinado, 2005), among other nitroreductase-like proteins. The minor nitroreductase NprB of R. capsulatus (Pérez-Reinado, 2005) and other putative uncharacterized nitroreductases of Escherichia coli and Salmonella (YdjA proteins) are included in the subfamily B2.

In Escherichia coli, the nfsA and nfsB genes code for the 27 and 24 kDa flavoproteins that are known as the major and minor oxygen-insensitive nitroreductases, respectively. NfsA uses NADPH to reduce nitrofurazone (Fig. 1) and other nitro-compounds, whereas NfsB uses both NADH and NADPH (Bryant et al., 1981; Zenno et al., 1996a, b, c, 1998a; Whiteway et al., 1998). Despite some biochemical similarities, NfsA and NfsB share very low identity with each other at the level of amino acids sequence, and they belong to different groups of nitroreductases (Fig. 4). An additional uncharacterized protein of Escherichia coli, the ydjA gene product (AAC74835), also shows similarity with other oxygen-insensitive bacterial nitroreductases that may constitute a different subgroup within the group B nitroreductases (Fig. 4). The nitroreductase NfsB of Escherichia coli is the most recognized enzyme used in cancer therapy because it converts the inert prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954; Fig. 1) into potent alkylating hydroxylamino nitrobenzamide derivatives. The expression of the Escherichia coli NfsB protein in human tumor cells increases their sensitivity to this chemical about 2500-fold. For this reason, this enzyme has been well studied at both structural and kinetics levels. The Escherichia coli NfsB nitroreductase shows apparent Km values for NADH and nitrofurazone of 6 and 64 μM, respectively, although these values increase 10-fold when they are measured at high concentrations of both substrates (Race et al., 2005). Recently it has been reported that this Escherichia coli nitroreductase catalyzes the bioconversion of CL-20 or 2,4,6,8,10,12-hexanitro-2,4,6,8,10,12-hexaazaisowurtzitane, a polycyclic nitramine explosive with a rigid caged structure that may be also degraded by Pseudomonas and Agrobacterium strains (Bhushan et al., 2003, 2004a, b; Trott, 2003). The nitro-compound hexahydro-1,3,5-trinitro-1,3,5-triazine or RDX (Fig. 1), another cyclic nitramine high energy compound, is also reductively transformed by an enteric oxygen-insensitive nitroreductase (Kitts et al., 2000).

The nitroreductase NR from Enterobacter cloacae is a homodimer, although it is also active as a 27 kDa monomer, and shows apparent Km values for TNT and NADPH of 56 and 443 μM, respectively (Bryant & DeLuca, 1991; Koder & Miller, 1998). Sequence analysis of the gene coding for the NR nitroreductase of Enterobacter cloacae (Bryant et al., 1991) and the RNR nitroreductase gene from an Enterobacter strain isolated from a patient (Koder et al., 2001) indicates that these nitroreductases are closely related to the Escherichia coli NfsB nitroreductase (Fig. 4).

First genetic studies carried out with the so-called classical nitroreductase Cnr of Salmonella enterica (typhimurium) were based on the generation of a genomic library and selection of colonies sensitive to 2-aminofluorene (Watanabe et al., 1989, 1990). The purification of the Salmonella Cnr nitroreductase demonstrated that it is a flavoenzyme similar to the minor nitroreductase NfsB of Escherichia coli. A defective Cnr nitroreductase with a Leu/Arg substitution in the putative FMN-binding site shows low affinity for the flavin cofactor and is completely devoid of nitroreductase activity, which is recovered by FMN addition (Watanabe et al., 1998). A second nitroreductase (SnrA) from Salmonella enterica that shows 87% amino acid sequence identity with the major nitroreductase NfsA of Escherichia coli has also been characterized. Like NfsA, SnrA is a FMN-containing homodimeric protein (28 kDa each monomer) that uses NADPH, but not NADH, as electron donor (Nokhbeh et al., 2002).

Several nitroreductases are present in Klebsiella sp. C1, and one of them, the NAD(P)H-nitroreductase I, has been purified and characterized (Kim et al., 2003; Kim & Song, 2005). The enzyme shows apparent Km values for TNT and NADPH of 64 and 358 μM, respectively, similar to those reported for other nitroreductases from enteric bacteria (Kim & Song, 2005). However, the N-terminal amino acid sequence of this protein shows low similarity with enteric nitroreductases. In contrast to other nitroreductases, that preferably reduce the nitro groups in the para position of TNT (Esteve-Núñez, 2001), the Klebsiella enzyme is able to reduce the ortho isomers to the corresponding hydroxylamino and amino derivatives. In addition, the enzyme does not reduce the nitro group in the para position of 2,4-dinitrotoluene, and only the ortho nitro group is reduced to generate 2-hydroxylamino-4-nitrotoluene (Kim & Song, 2005).

Two different nitroreductases, PnrA and PnrB, have been identified in the TNT-degrading strain P. putida JLR11. Both pnrA and pnrB genes are expressed constitutively, and sequence analysis suggests that PnrA and PnrB belong to the nitroreductase groups A and B, respectively (Fig. 4; Caballero et al., 2005b). Purified PnrA reduces TNT to 4-hydroxylamino-2,6-dinitrotoluene (4HADNT) with apparent Km values for NADPH and TNT of 20 and 5 μM, respectively. The NADPH-dependent nitrobenzene reductase of Pseudomonas pseudoalcaligenes JS45 catalyzes the four-electron reduction of nitrobenzene to hydroxylaminobenzene (Fig. 1). The low Km for nitrobenzene (5 μM) and the fact that the enzyme is induced by this nitro-compound suggest that nitrobenzene may be the physiological substrate for the enzyme (Somerville et al., 1995). However, this flavoenzyme also reduces 4-nitrobiphenyl ether to 2-amino-5-phenoxyphenol (Nadeau & Spain, 2000) and TNT to 4HADNT and other hydroxylamino intermediates (Fiorella & Spain, 1997).

Nitroreductases have also been found in some other heterotrophic bacteria, such as Thermus thermophilus (Park et al., 1992), Comamonas acidovorans (Groenewegen et al., 1992), Selenomonas ruminantium (Anderson et al., 2002), Vibrio fischeri (Zenno et al., 1994) and Vibrio harveyi (Lei et al., 1994). The NAD(P)H:FMN oxidoreductase FRaseI of Vibrio fischeri is a flavoprotein homologous to Escherichia coli NfsB nitroreductase, whereas the Vibrio harveyi NADPH-dependent flavin reductase FRP is homologous to Escherichia coli NfsA (Fig. 4). Although the wild-type proteins NfsB and NfsA of Escherichia coli do not reduce FMN, single amino acid substitutions convert these nitroreductases into flavin reductases, indicating that a small number of residues are responsible for substrate specificity (Zenno et al., 1996c, 1998a). Sequence and structural studies also confirm that NfsA and FRP belong to the NADPH-specific nitroreductase group A whereas NfsB and FRaseI belong to the NAD(P)H-nitroreductase group B (Tanner et al., 1996; Koike et al., 1998).

Both oxygenic and anoxygenic phototrophic bacteria also contain different nitroreductases. The FMN-containing DrgA protein of the cyanobacterium Synechocystis sp. PCC6803, which is similar to bacterial nitroreductases of the group B (Fig. 4), shows NAD(P)H-dependent nitroreductase activity with nitrobenzene, dinoseb and nitrofurazone as substrates, and also has flavin reductase and ferric reductase activities (Matsuo et al., 1998; Takeda et al., 2007). The anoxygenic phototrophic bacterium Rhodobacter capsulatus E1F1 reduces 2,4-dinitrophenol (Fig. 1) to 2-amino-4-nitrophenol by a NAD(P)H-dependent dinitrophenol reductase which shows apparent Km values for NADPH and 2,4-dinitrophenol of 37 and 70 μM, respectively (Blasco & Castillo, 1992, 1993). This flavoenzyme is rapidly inactivated by blue light, probably because the FMN prosthetic group produces an excited flavin that oxidizes some functional groups required for enzyme catalysis (Blasco et al., 1995). Recently, the nprA and nprB genes that code for two different nitroreductases have been isolated from R. capsulatus B10 (Pérez-Reinado, 2005). Both NprA and NprB proteins share only 14% amino acid sequence identity with each other and belong to different subgroups within the group B of nitroreductases (Fig. 4). Expression of the nprA gene is induced by a wide range of nitroaromatic and nitroheterocyclic compounds, whereas nprB gene expression is constitutive. Mutational analysis indicates that both gene products are involved in 2,4-dinitrophenol reduction, although a residual nitroreductase activity is found in the double mutant strain, suggesting the presence of additional nitroreductase(s) in this phototrophic bacterium (Pérez-Reinado, 2005).

Different nitroreductases have also been characterized in gram-positive bacteria, like Mycobacterium (Rafii et al., 1994, 2001), Staphylococcus (Streker et al., 2005), Clostridium (Kutty & Bennett, 2005) and Bacillus (Zenno et al., 1998b; Anlezark et al., 2002) species. The NfrA1 (YwcG) protein of Bacillus subtilis is an NADPH-dependent, FMN-containing oxidoreductase of 29 kDa that is induced by oxidative stress (Zenno et al., 1998b; Mostertz et al., 2004). This enzyme is homologous to the Staphylococcus aureus NfrA and the Escherichia coli NfsA proteins. However, the ywrO gene product of B. subtilis is a FMN-containing nitroreductase that is not homologous to the classical nitroreductases from enteric bacteria, but it is similar to the DT-diaphorase from Walker cells and other putative nitroreductases from mammals (Anlezark et al., 2002). The YwrO protein is able to reduce the prodrug CB1954, but in contrast to the Escherichia coli NfsB, which reduces this substrate to a mixture of the isomers 2HX [5-(aziridin-1-yl)-2-hydroxylamino-4-nitrobenzamide] and the more toxic 4HX [5-(aziridin-1-yl)-4-hydroxylamino-2-nitrobenzamide], the B. subtilis YwrO protein only produces the highly toxic 4HX isomer. The apparent Km of this nitroreductase for CB1954 is lower than the Km value of the NfsB enzyme for the same substrate, which is also desirable for tumor therapy techniques. Bacillus amyloliquefaciens possesses a putative ywrO ortholog, and two other additional nitroreductases, which are uncharacterized till date (Anlezark et al., 2002). In Clostridium acetobutylicum, two nitroreductases NitA and NitB involved in the transformation of 2,4-dinitrotoluene and TNT have been characterized, although NitA is more effective than NitB in TNT reduction (Kutty & Bennett, 2005). Both proteins are flavoenzymes with biochemical characteristics similar to those of the Escherichia coli NfsA nitroreductase. Curiously, the NitB protein shows a high amino acid sequence similarity (about 60%) to putative uncharacterized NAD(P)H-nitroreductases from the archaea Methanothermobacter thermoautotrophicus and Archaeoglobus fulgidus (Kutty & Bennett, 2005).

Structure and physiological function of bacterial nitroreductases

The way by which enzymes have acquired or lost their catalytic activities during evolution is a central matter in the study of protein structure and function. The physiological role of nitroreductases is still an open question to be answered, because although some representative bacterial nitroreductases have been characterized both biochemically and structurally, their real physiological functions are presently unknown. The crystal structures of the Escherichia coli NfsA and V. harveyi FRP group A nitroreductases, and the Escherichia coli NfsB, Enterobacter cloacae NR and V. fischeri FRaseI group B nitroreductases have been determined (Tanner et al., 1996; Koike et al., 1998; Parkinson et al., 2000; Kobori et al., 2001; Lovering et al., 2001; Haynes et al., 2002; Race et al., 2005). Three-dimensional structures of selected point mutants of the NfsB nitroreductase at positions that result in increased sensitization to the CB1954 prodrug have also been determined (Grove et al., 2003; Race et al., 2007). These studies, together with amino acid sequence comparisons, reveal that nitroreductases are globular proteins with conserved domains for FMN binding and for the interactions with the NAD(P)H electron donor and the nitroaromatic substrates (Fig. 5). These structural studies are also consistent with the ping–pong bi–bi catalytic mechanism of the enzyme, and provide an explanation of the broad substrate specificity of nitroreductases, establishing the basis for the improvement of the enzyme activity for medical applications (Johansson et al., 2003).

Figure 5

Structure of the Escherichia coli NfsB nitroreductase. (a) View looking down the non-crystallographic (molecular) twofold axis. (b) Dimer rotated 90° from the view in (a). The α-helices (red) are labeled from A to K and β-strands (yellow) are represented from 1 to 5. FMN and nicotinic acid ligand are coloured. From Lovering (2001).

Although with significant differences, mainly between proteins belonging to the NfsA/FRP and NfsB/FRaseI groups A and B (Fig. 4), the bacterial nitroreductases share a dimeric structure, with extensive dimer interface and a characteristic α+β-fold of the subunits (Fig. 5). The central hydrophobic core consists of five-stranded β-sheets surrounded by α-helices. The FMN prosthetic groups are bound in deep pockets at the dimer interface and interact with residues of both monomers, forming hydrogen bounds to one subunit and hydrophobic contacts to both. This set of interactions is well conserved in all nitroreductases and involves identical or similar residues (Koike et al., 1998; Parkinson et al., 2000; Kobori et al., 2001; Lovering et al., 2001; Haynes et al., 2002). The nicotinamide ring of NAD(P)H is placed between the flavin isoaloxazine ring and the conserved Phe124 residue, as deduced from the position in the NfsB protein of the analogous ligand nicotinic acid (Lovering et al., 2001; Haynes et al., 2002). Asn71Ser mutation in the Escherichia coli NfsB protein affects interactions with FMN whereas mutations Thr41Leu and Phe124Lys or Phe124Asn affect interactions with nicotinic acid. These mutations increase the NfsB activity with the CB1954 prodrug (Grove et al., 2003; Race et al., 2007). Also, substitution of the Phe124 residue by different unnatural amino acids improves the NfsB nitroreductase activity with CB1954 about 30-fold over the native residue, and more than twofold over the best possible natural amino acid (Jackson et al., 2006). The broad substrate specificity is explained by the inherent plasticity of the helix containing the Phe124 residue (helix H6 in Enterobacter cloacae NR and helix F in Escherichia coli NfsB), which shows an elevated variability in position for accommodating substrates of different sizes. In addition to the Phe124 residue, other nearly aromatic residues (Tyr68, Phe70 and Tyr123) may participate in substrate binding, and these residues vary among nitroreductases, suggesting that they may play a role in determining the substrate specificity of the enzymes (Lovering et al., 2001; Haynes et al., 2002). Surprisingly, in the crystal structure of the oxidized NfsB-nitrofurazone complex, this nitro-compound is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. Thus, the orientation of bound nitrofurazone may depend upon the redox state of the enzyme. The charge distribution on the FMN rings, which alters upon reduction, could be a key determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity (Race et al., 2005). Also, it has been described that substitution Leu33Arg greatly diminishes binding of FMN to the nitroreductase of Salmonella typhimurium (Watanabe et al., 1998).

Most nitroaromatic compounds are xenobiotic chemicals and it is believed that these compounds are not the physiological substrates of nitroreductases because they have been released into the environment by human activities only very recently. However, under the selective pressure of environmental pollution, the microbial capacity for degradation of xenobiotic chemicals may evolve (Timmis & Pieper, 1999; Rieger et al., 2002). Probably, the capability of some microorganisms to use a wide range of nitro-compounds may be due to the acquisition of mutations at the active site of the nitroreductases. Consequently, the primitive physiological function of these enzymes could be lost or modified allowing the reduction of different nitroaromatic and nitroheterocyclic compounds. Bacteria able to deal with these chemicals have a selective advantage and may survive in polluted environments (Timmis & Pieper, 1999). As mentioned, putative nitroreductase genes are present in archaea and both gram-negative and gram-positive bacteria. Genes required for degradation of xenobiotics may be recruited by various horizontal transfer mechanisms (Johnson & Spain, 2003), so that lateral gene transfer could be involved in the broad distribution of nitroreductases in prokaryotic organisms. Transmissible, plasmid-borne nitroreductase genes have been reported in different bacteria (Park & Kim, 2000) and in the conjugative transposon TnB1230, in which the new tetracycline resistant gene tet(W) of Butyrivibrio fibrisolvens is flanked by directly repeated sequences coding for putative oxygen-insensitive nitroreductases (Melville et al., 2004). It has also been hypothesized that the nitroreductase-like sequences present in the genomes of several protozoan species have been acquired by lateral gene transfer (Marques de Oliveira et al., 2007).

Different tentative physiological functions have been suggested for bacterial nitroreductases (Table 2). Because these enzymes have broad substrate specificity and reduce flavins, quinones and many different nitroaromatic and nitroheterocyclic compounds, it may be assumed that they play a role in general detoxification. However, some of these enzymes seem to be specialized in the transformation of a specific nitroaromatic compound because they are encoded by genes located in gene clusters or operons involved in particular degradative pathways. Thus, nitrobenzene reductase, chloronitrobenzene nitroreductase, nitrophenol reductase, dinitrophenol reductase and nitrobenzoate reductase are nitroreductases that play a specific role in productive catabolic pathways. In P. putida, the gene cluster encoding the enzymes required for 4-chloronitrobenzene degradation includes a chloronitrobenzene nitroreductase and it has been proposed that this enzyme has evolved to use chloronitrobenzene as the preferred substrate (Xiao et al., 2006). In P. fluorescens, the 2-nitrobenzoate reductase nbaA gene is also clustered together with genes involved in the nitrobenzoate degradative pathway, like the 2-hydroxylaminobenzoate mutase nbaB gene and the nbaY gene that codes for a chemoreceptor required for 2-nitrobenzoate chemotaxis (Iwaki et al., 2007).

View this table:
Table 2

Proposed physiological functions of oxygen-insensitive bacterial nitroreductases

FunctionsExperimental dataBacteria and nitroreductase namesReferences
Specific degradation pathwaysLow Km for nitroaromatic substrates. Nitroreductase gene located in degradative operons or gene clustersPseudomonas pseudoalcaligenes NbzA Pseudomonas putida CnbA Pseudomonas fluorecens NbaASomerville et al. (1995)Xiao et al. (2006) Iwaki et al. (2007)
Quinone-mediated reduction of extracellular azo-dyes and xenobioticsQuinone reductase activity and lawsone-dependent azo reductase activityEscherichi coli NfsA and NfsBRau & Stolz (2003)
Response to oxidative stress and cellular redox balancingInduction by paraquat and oxidative stress conditions. Control by the SoxRS systemEscherichia coli NfsA Rhodobacter capsulatus NprA Bacillus subtilis NfrA Staphylococcus aureus NfrALiochev et al. (1999) Pérez-Reinado (2005) Mostertz et al. (2004) Streker et al. (2005)
Disulfide stress responseDisulfide reductase activityStaphylococcus aureus NfrAStreker et al. (2005)
Supply of reduced flavins for the luciferase reaction (bioluminescence)Flavin reductase activityVibrio fischeri FRase I Vibrio harveyi FRPLei et al. (1994) Zenno et al. (1994) Zenno et al. (1998a)
Iron metabolismFerric reductase activity and Fenton reaction catalysisSynechocystis sp. DgrATakeda et al. (2007)
Chromate reductionChromate reductase activityEscherichia coli NfsA and NfsB Vibrio harveyi FRPAckerley et al. (2004) Kwak et al. (2003)
Lipoamide dehydrogenaseN-terminal sequence similarityMycobacterium sp. Pyr-1Rafii et al. (2001)
Dihydropteridine reductaseN-terminal sequence similarity. Dihydropteridine reductase activity in vitroEscherichia coli NfsB Rhodobacter capsulatus NprAVasudevan et al. (1988) Pérez-Reinado et al. (2008)
Cobalamin (vitamin B12) biosynthesisSynthesis of B12 impaired in mutants defective in the bluB gene coding for a nitroreductase-like proteinRhodobacter capsulatus BluB Sinorhizobium meliloti BluB Selenomonas ruminantium BluBPollich & Klug (1995) Campbell et al. (2006) Anderson et al. (2002)

Lawsone (2-hydroxy-1,4-napthoquinone) and other quinones are redox mediators in some biodegradative processes, like anaerobic reduction of azo compounds used as dyes for textiles, food and cosmetics (Field et al., 2000; Rau & Stolz, 2003). The quinones are enzymatically reduced in the cells and then, the resulting hydroquinones chemically reduce the azo dyes outside the cells. Both NfsA and NfsB Escherichia coli nitroreductases have been shown to function, under anaerobic conditions, as lawsone-dependent azo reductases. This activity enables Escherichia coli cells to reduce anaerobically different sulfonated and polymeric azo compounds (Rau & Stolz, 2003). Therefore, nitroreductases could also be involved in the pathways for the transfer of reducing power from bacterial cells to the various natural and xenobiotic compounds present in the extracellular media (Rau & Stolz, 2003). It is worth noting that this electron transfer pathway via nitroreductase could also serve as a mechanism to dissipate excess reducing power for redox balancing.

It is also described that nitroreductases may participate in the response to oxidative stress. The major nitroreductase NfsA of Escherichia coli is regulated by the SoxRS system, which is involved in the control of the oxidative stress response (Liochev et al., 1994, 1999). Also, the R. capsulatus NprA nitroreductase is induced by paraquat (Fig. 1), a superoxide-generating agent, and at low NAD(P)H/NAD(P)+ ratios (Pérez-Reinado et al., 2005), and the NfrA nitroreductases from Staphylococcus aureus and Bacillus subtilis are induced under oxidative stress conditions (Mostertz et al., 2004; Streker et al., 2005). The Staphylococcus aureus NfrA protein also exhibits a weak disulfide reductase activity and it has been proposed that is involved in the control of the cellular redox balance and the thiol-disulfide stress response (Streker et al., 2005).

Nitroreductase-like proteins have been involved in the bioluminescent process, catalyzing the reduction of FMN by NAD(P)H to provide the reduced form of the flavin required for the luciferase reaction (Lei et al., 1994). As mentioned, the NAD(P)H flavin oxidoreductase (FRaseI) of the luminescent bacterium Vibrio fischeri and the Escherichia coli NfsB nitroreductase are closely related (Fig. 4), and have similar structural and biochemical characteristics. FRaseI uses FMN as the main electron, but also shows nitroreductase activity (Zenno et al., 1994). Curiously, a single amino acid change at the active site of the Escherichia coli NfsB protein converts this nitroreductase to a flavin reductase with similar properties to the V. fischeri FRaseI, although the specific activity of NfsB is three fold higher than the activity exhibited by the FRaseI (Zenno et al., 1996c). Similarly, a single substitution in the polypeptide chain of Escherichia coli NfsA converts this enzyme to a flavin oxidoreductase similar to the NADPH-dependent flavin reductase FRP of Vibrio harveyi (Zenno et al., 1998a).

Flavin reductases are able to catalyze ferric ion reduction, which in general depends on reduced flavins provided by flavin reductases (Fontecave et al., 1994). Very recently, it has been reported that the DrgA protein of the cyanobacterium Synechocystis sp. shows nitroreductase, quinone reductase, flavin reductase and ferric reductase activities, and also catalyzes the Fenton reaction that occurs when iron and hydrogen peroxide react to generate hydroxyl radicals (Takeda et al., 2007). In the presence of Fe2+, the the product of the ferric reductase activity, hydrogen peroxide is reduced through one electron transfer to generate the hydroxyl radical, a highly reactive oxygen species. In addition to EDTA and citrate, transferrin and ferritin may also act as substrates of the ferric reductase activity of DrgA, so that it has been proposed that DrgA plays a role in iron metabolism under physiological conditions and could also catalyze the Fenton reaction under highly reductive conditions, like exposure to strong light (Takeda et al., 2007).

It has been reported that some bacterial nitroreductases show chromate reductase activity (Kwak et al., 2003; Ackerley et al., 2004). The chromate reductase from Pseudomonas ambigua is highly similar to the Vibrio harveyi and Escherichia coli nitroreductases, and these enzymes also reduce Cr(VI)–Cr(III) with high affinity. The Vmax/Km values of these nitroreductases for chromate reduction suggest that the major nitroreductase NfsA of Escherichia coli is twice as efficient as the NfsB nitroreductase, whereas the V. harveyi nitroreductase is 12-fold as efficient as Escherichia coli NfsB (Kwak et al., 2003). For this reason, it has been proposed the utilization of bacterial nitroreductases for the remediation of chromate polluted environments (Kwak et al., 2003; Ackerley et al., 2004).

The nitroreductase Pyr-1 of Mycobacterium sp. seems to be a lipoamide dehydrogenase, as deduced from its N-terminal sequence (Rafii et al., 2001). The N-terminal end of a dihydropteridine reductase isolated from Escherichia coli also shows a high identity with the N-terminus of the NfsB protein (Vasudevan et al., 1988). However, the physiological roles of nitroreductases as lipoamide dehydrogenase or dihydropteridine reductase have not yet been demonstrated. We have recently found that the purified NprA nitroreductase from R. capsulatus shows dihydropteridine reductase activity in vitro (Pérez-Reinado, 2008).

Selenomonas ruminantium contains a flavoprotein that is probably involved in the metabolism of the coenzyme B12 (cobalamin), a cofactor required for anaerobic fermentative metabolism and also for the synthesis of methionine and deoxynucleotides in some bacteria. This flavoenzyme is a homodimer of 45 kDa, contains FMN as cofactor and is homologous to bacterial nitroreductases, but its nitroreductase activity was not detected (Anderson et al., 2002). The nitroreductase-like BluB protein has been implicated in the biosynthesis of cobalamin in Rhodobacter capsulatus (Pollich & Klug, 1995), and more recently in Sinorhizobium meliloti (Campbell et al., 2006). In this symbiotic nitrogen fixing bacterium, the BluB flavoprotein is required for the production of 5,6-dimethylbenzimidazole, the lower ligand of B12 (Campbell et al., 2006). Recently, it has been demonstrated that BluB triggers an unusual fragmentation of the FMN cofactor to form the 5,6-dimethylbenzimidazole ligand of B12 (Taga et al., 2007).

It seems evident that this puzzling situation regarding the physiological roles of bacterial nitroreductases needs to be rationalized. First, these enzymes are widely distributed among microorganisms, and most of them contain several nitroreductase-like flavoproteins, which is a clear evidence of the relevance of these proteins in the microbial metabolic activities. Second, the biochemical and kinetic studies reveal that nitroreductases catalyze the NAD(P)H-dependent reduction of a wide variety of substrates, including nitroaromatic and nitroheterocyclic compounds, quinones, flavins, as well as some ions like Fe3+ and CrO42−. The three-dimensional structure analysis reveals an inherent plasticity of the active site for the substrate binding and variability in the amino acid residues participating in these interactions, thus explaining the different reductase activities for nitroaromatics, quinones, flavins and other compounds. Third, gene duplications, lateral gene transfer and accumulation of different mutations at specific sites have created a set of nitroreductase-like proteins that perform a wide variety of functions in the different organisms. The versatility and multiplicity of these enzymes have probably allowed the specialization of some nitroreductases in different metabolic functions, with or without loss of the other reductase activities. Thus, some enzymes are specifically associated to metabolic pathways or reductive degradation routes of different nitroaromatic compounds whereas other nitroreductase-like proteins may be involved in other precesses, such as bioluminescence, coenzyme B12 biosynthesis, oxidative stress response or redox balancing. Bacteria containing nitroreductase enzymes with broad sustrate specifity probably have adaptative or selective advantages to survive in natural environments. The versatility of this group of enzymes also constitutes the basis of their use in different biotechnological applications.

Regulation of nitroreductase gene expression

Studies on nitroreductases are mainly focused on kinetics mechanisms and substrate specificity, particularly in the aspects related to their effectiveness with the prodrugs used in cancer therapies or with resistance to antibiotics, but studies on regulatory aspects are scarce.

The first studies on nitroreductase gene regulation were carried out with the Escherichia coli nfsA gene. The NfsA nitroreductase is induced in the presence of the herbicide paraquat (Liochev et al., 1994, 1999; Paterson et al., 2002). This compound generates the superoxide anion by reduction of molecular oxygen. This type of oxidative stress is mainly generated in the reduction, through one electron, of the aromatic nitro groups, quinones and azo-dyes. The regulatory system that detects and responds to this oxidative stress is encoded by the soxRS genes. Therefore, it has been proposed that the nfsA gene is a member of the soxRS regulon, which could contribute to the protection against oxidative stress minimizing the redox cycling processes (Liochev et al., 1999; Paterson et al., 2002). A similar study was carried out with the Escherichia coli nfsB gene, but in this case a minimal induction with paraquat was observed (Liochev et al., 1999).

Macroarray studies have also demonstrated that Escherichia coli nfsA gene is a member of the mar regulon (Barbosa & Levy, 2000). In Escherichia coli, the MarRA system controls the response to different antibiotics and environmental pollutants. This regulatory system is related to the SoxRS system, and in fact, both regulatory proteins MarA and SoxS, and also the constitutively expressed protein known as Rob (Skarstad et al., 1993), may bind to a similar consensus sequence termed mar/sox/rob box, which is located in the promoter regions of the different target genes. This box consists of the 20-bp sequence: ATNGCACNNWNNRYYAAAYN, with Y=C or T, W=A or T, R=A or G, and N=any nucleotide (Martin et al., 1999, 2000). Some nucleotides of this sequence may change, but there are two highly conserved recognition elements, GCAC (RE1) and YAAA (RE2). These two elements seem to establish important links with the two helix–turn–helix motives of the MarA, SoxS, and Rob regulatory proteins (Li & Demple, 1996; Rhee et al., 1998; Gillette et al., 2000; Griffith & Wolf, 2001). The Escherichia coli nfsA gene and the adjacent rimK and ybjC genes constitute an operon regulated by both mar and sox genes (Barbosa & Levy, 2000; Paterson et al., 2002), and it has also been demonstrated that activation of nfsB gene expression also takes place through MarA, which binds to an unusual mar box located in the promoter region of the nfsB gene (Barbosa & Levy, 2002).

The Escherichia coli soxRS regulon is composed of at least 17 related genes which respond to the redox status of the cell (Greenberg et al., 1990; Liochev et al., 1994; Ma et al., 1996; Aono et al., 1998). SoxR is a dimeric sensor with two [2Fe–2S] centers that detects oxidative stress and induces expression of soxS gene, which encodes a positive transcriptional regulator belonging to the AraC/XylS family that, in turn, induces expression of the genes of the sox regulon (Amábile-Cuevas & Demple, 1991; Wu & Weiss, 1992; Li & Demple, 1994; Ding et al., 1996; Pomposiello et al., 2001). When Escherichia coli cells are subjected to oxidative stress by superoxide anion or paraquat, or when the intracellular NAD(P)H to NAD(P)+ ratio decreases, the iron–sulfur centers of SoxR become oxidized, and this is the active form of the protein that induces soxS gene expression (Fig. 6). SoxS is intrinsically unstable, and after cessation of the oxidative stress is quickly degraded. Protein–DNA and protein–protein interactions seem to be of great importance to enhance SoxS stability (Shah & Wolf, 2006). Therefore, the SoxRS system differs from the most common bacterial two-component regulatory systems in that the SoxS protein required for expression of the target genes is not posttranslationally modified, but instead, is synthesized de novo in response to the oxidation of the [2Fe–2S] clusters of the SoxR sensor that convert this protein into a functional transcription activator of soxS gene expression. Then, the newly synthesized, intrinsically unstable SoxS activates the transcription of the genes of the sox regulon for protection against oxidative stress (Shah & Wolf, 2006).

Figure 6

The bacterial SoxRS and MarRA regulatory systems. In Escherichia coli, the SoxRS system protects against the oxidative stress induced by superoxide anion or nitric oxide. The presence of paraquat and the existence of a low intracellular NAD(P)H/NAD(P)+ ratio also induce expression of the genes of the sox regulon. The MarRA system acts in response to the presence of several antibiotics and environmental contaminants. Weak acids, such as salicylic acid or 2,4-dinitrophenol, bind to the repressor MarR and cause its inactivation, thus allowing the transcription of the marA gene. MarA is an activator that, in turn, allows induction of the genes controlled by this system (see text for details).

The resistance to multiple antibiotics in Escherichia coli is associated with the mar locus. Expression of the mar genes also increases resistance to organic solvents and environmental pollutants, and activates genes related to oxidative stress (Martin et al., 1996; White et al., 1997). The mar locus is organized into two divergent transcriptional units, marC and marRAB (Fig. 6). The expression of both transcriptional units is under the control of the same operator, marO, which is located between marC and marR genes (Martin et al., 1996; Sulavik et al., 1997). In the marRAB operon, the product of the first gene (marR) represses mar gene transcription, whereas the product of the second gene (marA) stimulates its expression (Cohen et al., 1993a; Ariza et al., 1994). MarR repressor binds as a dimer to the promoter mar region in two sites, the first between the −35 and −10 region, and the second in the ribosome-binding site of the marR gene (Fig. 6). MarR interacts directly with aromatic and nitroaromatic compounds, like salicylic acid and 2,4-dinitrophenol, and also with plumbalgin (a quinone that causes oxidative stress). This interaction decreases the affinity of MarR for these binding sites (Cohen et al., 1993b; Martin & Rosner, 1995; Alekshum & Levy, 1999). MarA is a transcriptional activator of the AraC/XylS family that shows about 50% amino acid sequence identity to SoxS and binds to the main mar box located in the marO region, upstream from the −35 box, and probably also to an additional mar box present in the marO region because its deletion causes a decrease in marRAB transcription (Martin et al., 1996). MarA is able to activate the expression of more than 60 genes by its binding to the mar boxes of the different target genes (Martin et al., 1996, 2000).

The different promoters of the genes activated by the MarA, SoxS and Rob regulatory proteins do not respond to these activators to the same degree, so that the cells may respond specifically to the different stresses (Ariza et al., 1995; Jair et al., 1996b; Martin et al., 1996; Griffith & Wolf, 2001). These regulatory proteins interact with the RNA polymerase in two different ways, and depending on this fact, there are two types of promoters. In class I promoters, the mar/sox/rob box is located at 14–16 or 26–27 bp upstream from the −35 position, usually with a reverse orientation in relation to the promoter. Activation of these promoters requires interaction between MarA or SoxS and the C-terminal domain of the α subunit of the RNA polymerase. In class II promoters, the mar/sox/rob box is located inside the −35 region with a forward orientation and MarA or SoxS activation is not dependent on the interaction between the regulatory protein and the C-terminal domain of the α subunit of the RNA polymerase (Jair et al., 1996a). It has been demonstrated that SoxS binds to the Escherichia coli nfsA promoter in a manner characteristic of the class I promoter, although the mar/sox box is found in the forward orientation (Wood et al., 1999; Paterson et al., 2002).

MarRA/SoxRS-like regulatory systems are probably involved in the control of the nitroreductase gene expression in other bacteria. Transcriptome and proteome analyses have revealed that the MarR-type regulatory protein YodB of Bacillus subtilis is involved in the repression of the yodC gene, which codes for a nitroreductase that is induced by catechol and 2-methylhydroquinone (Van Duy, 2007). The identification of two putative mar/sox/rob boxes in the promoter region of the nprA gene of Rhodobacter capsulatus B10, that codes for the main nitroreductase of this phototrophic bacterium, and the induction of the nprA gene expression by salicylic acid and 2,4-dinitrophenol also suggest a role for the MarRA regulatory system in the control of this nitroreductase gene (Pérez-Reinado, 2005). Upregulation of the R. capsulatus nprA gene by paraquat or conditions that cause a decrease in the intracellular NAD(P)H to NAD(P)+ ratio also suggests that the nprA gene is involved in the response to oxidative stress conditions in this bacterium (Pérez-Reinado, 2005). In addition to Mar and Sox, other regulatory systems may also participate in the control of nitroreductase gene expression. Thus, dinitrophenol reduction in R. capsulatus is also regulated by the general nitrogen regulatory (Ntr) system, either directly or indirectly, by controlling expression of the Rnf proteins that are involved in the electron supply to both dinitrogen fixation and dinitrophenol reduction (Sáez, 2001).

Biotechnological applications of bacterial nitroreductases

The NAD(P)H-dependent bacterial nitroreductases have received special attention because they can be used for biodegradation and bioremediation of nitroaromatics (Spain et al., 1995; Rieger et al., 2002; Lewis et al., 2004; Ramos et al., 2005), as well as in biomedicine, particularly in several cancer therapies (Knox et al., 1993; Knox & Connors, 1995, 1997; Green et al., 2004; Searle et al., 2004). In addition, nitroreductases can be used in the production of intermediates for synthesis of comercially important materials. The nitrobenzene reductase from Pseudomonas pseudoalcaligenes JS45 catalyzes the conversion of a nitro group of nitrobenzene to the corresponding hydroxylamino derivative, but also reduces 4-nitrobiphenyl ether to 2-amino-5-phenoxyphenol. The biotechnological application relies on the use of o-aminophenols as starting materials for the synthesis of polybenzoxazols (Nadeau & Spain, 2000).

Bioremediation is the biotechnological application of organisms with degradative capabilities, or their enzymes, for removing pollutants from the environment. TNT is the most widely used nitroaromatic compound and there are many places world-wide polluted with this recalcitrant explosive (Hartter et al., 1985; Rieger et al., 2002; Lewis et al., 2004). Biodegradation of TNT has been investigated in several methanogens, clostridia, denitrifyers and sulfate-reducing bacteria, and even in the microbial communities of the bovine rumen, which is a highly reductive environment (Fleischmann et al., 2004). Some organisms that able to reduce the nitro substituents of TNT under anaerobic conditions are Methanococcus sp. (Boopathy et al., 1994; Boopathy & Kulpa, 1994), Desulfovibrio sp. (Boopathy & Kulpa, 1992), Clostridium acetobutylicum (Gorontzy et al., 1993; Khan et al., 1997), and P. putida JLR11 (Esteve-Núñez & Ramos, 1998; Caballero et al., 2005b). Different aerobic and anaerobic systems have been used for remediation of TNT and other polynitroaromatic compounds, including static pile and windrow composting, bioslurry, and phytoremediation (Funk et al., 1993; Smets et al., 1999; Hawari et al., 2000a; Peres & Agathos, 2000; Rodgers & Bunce, 2001; Zhang et al., 2001; Lewis et al., 2004; Schrader & Hess, 2004). Transgenic plants bearing bacterial nitroreductase genes, or combined treatments with both plants and microorganisms able to degrade nitroaromatic compounds, are expected to be used as efficient decontaminating procedures in the near future (French et al., 1999; Hannink et al., 2001, 2002; Kurumata et al., 2005). In addition, some efforts have been directed to develop new genetically modified bacterial strains able to degrade nitroaromatic compounds by hybrid pathways (Duque et al., 1993; Michán, 1997). Approaches for monitoring the bioremediation processes and their efficiency are also necessary (Frische et al., 2002, 2003; Frische & Hoper, 2003). Biosensors based on living microorganisms, enzymes or immunochemical reactions may also be developed to detect sites and materials polluted with nitroaromatic compounds (Ramos et al., 2005).

On the other hand, bacterial nitroreductases have also a great clinical interest. In the ADEPT technique for cancer therapy, a bacterial nitroreductase, usually the Escherichia coli NfsB protein, is conjugated to tumor cell-specific antibodies to direct the enzyme to the tumor. Then, an inert prodrug, such as the dinitrobenzamide CB1954 (Fig. 1), is converted by this nitroreductase into highly cytotoxic hydroxylamino products that kill both replicating and nonreplicating tumor cells (Anlezark et al., 1992; Knox et al., 1993; Knox & Connors, 1995, 1997). In the GDEPT or VDEPT techniques, the bacterial nitroreductase gene is delivered to the tumor cells using DNA complexes or viral vectors, thus allowing the synthesis of the enzyme in the tumor cells, which become sensitized to the prodrug (Grove et al., 1999, 2003; Green et al., 2004; Searle et al., 2004; Lukashev et al., 2005; Benouchan et al., 2006). Clostridia have also been used as a nitroreductase gene delivery vector (Theys et al., 2006). In the last few years, a number of potential substrates for the Escherichia coli NfsB nitroreductase, including different dinitrobenzamide mustards, oxazino-acridines and nitrobenzyl and nitroheterocyclic carbamate prodrugs, have been tested (Hay et al., 2003a, b; Asche & Kucklaender, 2006; Asche et al., 2006; Ouberai et al., 2006; Atwell et al., 2007). Site-directed mutagenesis and site-specific incorporation of unnatural amino acids at the active site of the NfsB protein has been performed to increase activity or to change substrate specificity with the main purpose of using new prodrugs (Grove et al., 2003; Jackson et al., 2006; Race et al., 2007). A phase I clinical trial of CB1954 and replication-defective, nitroreductase-expressing adenovirus is in progress in human patients with primary and secondary liver cancer, but prodrug dose-limiting hepatotoxicity has been observed (Searle et al., 2004; Tang et al., 2005). Recently it has been reported that the antitumor protection by adenoviral GDEPT using the nitroreductase/CB1954 system is significantly enhanced when the heat shock protein Hsp70 is coexpressed (Djeha et al., 2005; Lipinski et al., 2006). A microfluidic reactor for screening cancer prodrug activation using silica-immobilized NbzA nitrobenzene reductase from Pseudomonas pseudoalcaligenes JS45 has been performed (Berne et al., 2006). Immobilized nitroreductase is more stable than in solution and the packed enzyme in the microfluidic reactor is able to use nitrobenzene, CB1954 and the proantibiotic nitrofurazone (Fig. 1). Therefore, the flow-through system provides a rapid and reproducible screening method for determining the NbzA-catalyzed activation of prodrugs and proantibiotics (Berne et al., 2006). Nitroreductases are also responsible for the toxicity and the mutagenic and carcinogenic character of a large number of nitroaromatics, due to the formation of toxic nitroso and hydroxylamino derivatives, and this fact has also been exploited in the use of nitrofuran-derivatives as antibiotics (Beland et al., 1989; Whiteway et al., 1998; Race et al., 2005). Nitroreductase is also involved in the resistance to metronidazole (Fig. 1) developed by Helicobacter pylori, a bacterium that causes gastric ulcers and constitutes a risk factor against adenocarcinoma and gastric lymphoma (Goodwin et al., 1998; Kwon et al., 2000; Solcà, 2000; Han et al., 2007), and Bacteroides fragilis strains, the most common isolates from clinically significant anaerobic infections (Schapiro et al., 2004). In H. pylori, susceptibility to metronidazole results from its activation as a toxic agent by two different nitroreductases, RdxA, which is abundant in almost all metronidazole-sensitive clinical isolates, and FrxA, which is present at a low level in most of them. All metronidazole-resistant isolates have alterations in the RdxA and/or the FrxA proteins (Han et al., 2007). The fdxA gene, which codes for a [4Fe–4S]-type ferredoxin, can down-regulate the expression of the frxA nitroreductase gene (Mukhopadhyay et al., 2003). Application of the Escherichia coli nitroreductase for the elimination of human T cells with metronidazole has also been reported (Verdijk et al., 2004). Targeted ablation of different cell lines by the bacterial nitroreductase-prodrug system may constitute a new tool for cell regeneration studies, for determining the function of cell populations in vivo and for screening abnormalities and cellular degeneration (Curado et al., 2007; Kwak et al., 2007; Pisharath et al., 2007). On the other hand, the Ames test to predict the mutagenic character of a chemical also exploits the nitroreductases of Salmonella strains (McCoy et al., 1981; Yamada et al., 1997; Salamanca-Pinzón, 2006). More sensitive, new tester strains have been constructed by introducing plasmids that encode the major and minor nitroreductases genes, nfsA and nfsB, of Escherichia coli in different combinations with additional genes (Carroll et al., 2002).

Concluding remarks and perspectives

Bioremediation is an effective treatment for decontamination of worldwide areas polluted with polynitroaromatic compounds, such as those sites where explosives, pesticides, dyes, solvents or some plastics are manufactured or handled. In this regard, many studies on physiology, biochemistry and genetics of the organisms that can be potentially used in these biotreatments have been performed in the last decades, including the search for enzymes involved in the degradation of nitroaromatic compounds. Among these enzymes, the bacterial nitroreductases that catalyze the sequential transfer of electron pairs to the nitro groups of the aromatic compounds, forming the nitroso, hydroxylamino and amino derivatives, are of special biotechnological interest. In addition to the bioremediation and biocatalysis approaches, bacterial nitroreductases may be used in different medical applications, including cancer therapies based on the reduction of an inert prodrug to produce highly cytotoxic hydroxylamino intermediates in the sensitized tumor cells. Nearly all these studies have been carried out with the Escherichia coli NfsB nitroreductase, or its nfsB gene, and the prodrug CB1954, which shows high selectivity in different nitroreductase-transfected cell lines, such as human ovarian, colorectal and pancreatic. However, the potential application of emergent nitroreductases from many different bacterial species in these antitumor techniques remains unexplored. Nitroreductases may reduce a wide range of nitroaromatic and nitroheterocyclic compounds, so that future works may deal with the search for new potential substrates and prodrugs. Different bacterial nitroreductases have been purified, and their biochemical and kinetic parameters have been characterized. In addition, the crystal structures of several nitroreductases have been solved. However, it is worth noting that the physiological role(s) of most nitroreductases enzymes remains unclear, although it can not be discarded the participation of some nitroreductases in different metabolic processes due to their substrate versatility. Future efforts directed towards the elucidation of the real physiological function(s) of these enzymes may be crucial to understand both enzyme evolution and catalysis. Finally, studies on regulation of nitroreductase gene expression, which in Escherichia coli and R. capsulatus seem to be controlled by the MarRA and SoxRS regulatory systems in response to several antibiotics, environmental contaminants and specific oxidative stress conditions, might also reveal differences between bacteria and will contribute to a better understanding of the in vivo function and the potential biotechnological applications of these enzymes with huge environmental and clinical interest. In this sense, genomic and metagenomic approaches will probably contribute in the near future to find new organisms and enzymes able to degrade polynitroaromatic compounds and to obtain more information about bacterial nitroreductases for developing new biotechnological applications.


This work was funded by Ministerio de Ciencia y Tecnología (Grants BMC 2002-04126-CO3-03 and BIO2005-07741-CO2-01) and Junta de Andalucía (Grant CVI-0117). E.P.R. was recipient of a fellowship from the Ministerio de Ciencia y Tecnología. M.D.R. is a postdoctoral research associate holding a contract from Junta de Andalucía, Spain. The authors thank Dr J.A. Cole for helpful advice and suggestions. The fruitful collaboration of GEMASUR, S.L. is also acknowledged.


  • Editor: Jiri Damborsky


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