Reduction of polynitroaromatic compounds: the bacterial nitroreductases.

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.

The NprA nitroreductase required for 2,4-dinitrophenol reduction in Rhodobacter capsulatus is a dihydropteridine reductase.

The Rhodobacter capsulatus nprA gene codes for a putative nitroreductase. A recombinant His(6)-NprA protein was overproduced in Escherichia coli and purified by affinity chromatography. This protein contained FMN and showed nitroreductase activity with a wide range of nitroaromatic compounds, such as 2-nitrophenol, 2,4-dinitrophenol, 2,6-dinitrophenol, 2,4,6-trinitrophenol (picric acid), 2,4-dinitrobenzoate and 2,4-dinitrotoluene, and with the nitrofuran derivatives nitrofurazone and furazolidone. NADPH was the main electron donor and the ortho nitro group was preferably reduced to the corresponding amino derivative. The apparent K(m) values of NprA for NADPH, 2,4-dinitrophenol, picric acid and furazolidone were 40 microM, 78 microM, 72 microM and 83 microM, respectively, at pH and temperature optima (pH 6.5, 30 degrees C). Escherichia coli cells overproducing the NprA protein were much more sensitive to the prodrug 5-(aziridin-1-yl)-2,4-dinitrobenzamide (CB1954) used in cancer therapy than non-transformed cells. NprA showed the highest activity with the quinonoid form of 6,7-dimethyl-7,8-dihydropterine as substrate, so that NprA may be involved in the synthesis of tetrahydrobiopterin in R. capsulatus. Expression of a transcriptional nprA-lacZ gene fusion was induced by phenylalanine or tyrosine, but not by other amino acids like glutamate or alanine. Furthermore, both nitroreductase activity and phenylalanine assimilation were inhibited in vivo by ammonium. A mutant defective in the nprA gene showed better growth rate with Phe or Tyr as nitrogen source than the wild-type strain, although both strains showed similar growth in media with Glu or without added nitrogen. These results suggest that the NprA nitroreductase may act in vivo as a dihydropteridine reductase involved in aromatic amino acids metabolism.

Towards a global analysis of porcine alveolar macrophages proteins through two-dimensional electrophoresis and mass spectrometry.

Alveolar macrophages (AM) are the primary phagocytes of the innate immune systems, constituting a link between innate and adaptive immunity. With the aim of studying the porcine AM biology and the dynamics of pig-pathogen cell interactions, we have obtained a reference 2-DE map of the porcine AM proteins. The proteins were separated by 2-DE using a 5-8 range pH gradient in isoelectric focusing and over 800 spots were detected. A set of proteins, covering the pI 5.2-7.4 and M(W) 19 to 106kDa ranges, was subjected to MS analysis and 106 proteins were assigned identification by PMF, this identification being confirmed by MS/MS. An important number of proteins is involved in immunological functions, signalling process, transport or apoptosis, confirming that macrophages are involved in a wide range of biological functions. This reference map provides a useful tool for identifying protein pattern changes as a result of inflammation, exposure to infectious agents or genetic diseases.

The EADGENE Microarray Data Analysis Workshop (open access publication).

Microarray analyses have become an important tool in animal genomics. While their use is becoming widespread, there is still a lot of ongoing research regarding the analysis of microarray data. In the context of a European Network of Excellence, 31 researchers representing 14 research groups from 10 countries performed and discussed the statistical analyses of real and simulated 2-colour microarray data that were distributed among participants. The real data consisted of 48 microarrays from a disease challenge experiment in dairy cattle, while the simulated data consisted of 10 microarrays from a direct comparison of two treatments (dye-balanced). While there was broader agreement with regards to methods of microarray normalisation and significance testing, there were major differences with regards to quality control. The quality control approaches varied from none, through using statistical weights, to omitting a large number of spots or omitting entire slides. Surprisingly, these very different approaches gave quite similar results when applied to the simulated data, although not all participating groups analysed both real and simulated data. The workshop was very successful in facilitating interaction between scientists with a diverse background but a common interest in microarray analyses.

Regulation and characterization of two nitroreductase genes, nprA and nprB, of Rhodobacter capsulatus.

Among photosynthetic bacteria, strains B10 and E1F1 of Rhodobacter capsulatus photoreduce 2,4-dinitrophenol (DNP), which is stoichiometrically converted into 2-amino-4-nitrophenol by a nitroreductase activity. The reduction of DNP is inhibited in vivo by ammonium, which probably acts at the level of the DNP transport system and/or physiological electron transport to the nitroreductase, since this enzyme is not inhibited by ammonium in vitro. Using the complete genome sequence data for strain SB1003 of R. capsulatus, two putative genes coding for possible nitroreductases were isolated from R. capsulatus B10 and disrupted. The phenotypes of these mutant strains revealed that both genes are involved in the reduction of DNP and code for two major nitroreductases, NprA and NprB. Both enzymes use NAD(P)H as the main physiological electron donor. The nitroreductase NprA is under ammonium control, whereas the nitroreductase NprB is not. In addition, the expression of the nprB gene seems to be constitutive, whereas nprA gene expression is inducible by a wide range of nitroaromatic and heterocyclic compounds, including several dinitroaromatics, nitrofuran derivatives, CB1954, 2-aminofluorene, benzo[a]pyrene, salicylic acid, and paraquat. The identification of two putative mar/sox boxes in the possible promoter region of the nprA gene and the induction of nprA gene expression by salicylic acid and 2,4-dinitrophenol suggest a role in the control of the nprA gene for the two-component MarRA regulatory system, which in Escherichia coli controls the response to some antibiotics and environmental contaminants. In addition, upregulation of the nprA gene by paraquat indicates that this gene is probably a member of the SoxRS regulon, which is involved in the response to stress conditions in other bacteria.