The role of the NADH-dependent nitrite reductase, Nir, from Escherichia coli in fermentative ammonification.

Nitrate and nitrite reduction are of paramount importance for nitrogen assimilation and anaerobic metabolism, and understanding the specific roles of each participating reductase is necessary to describe the biochemical balance that dictates cellular responses to their environments. The soluble, cytoplasmic siroheme NADH-nitrite reductase (Nir) in Escherichia coli is necessary for nitrate/nitrite assimilation but has also been reported to either "detoxify" nitrite, or to carry out fermentative ammonification in support of anaerobic catabolism. Theoretically, nitrite detoxification would be important for anaerobic growth on nitrate, during which excess nitrite would be reduced to ammonium. Fermentative ammonification by Nir would be important for maximization of non-respiratory ATP production during anaerobic growth in the presence of nitrite. Experiments reported here were designed to test the potential role of Nir in fermentative ammonification directly by growing E. coli along with mutant strains lacking Nir or the respiratory nitrite reductase (Nrf) under anaerobic conditions in defined media while monitoring nitrogen utilization and fermentation metabolites. To focus on the role of Nir in fermentative ammonification, pH control was used in most experiments to eliminate nitrite toxicity due to nitric acid formation. Our results demonstrate that Nir confers a significant benefit during fermentative growth that reflects fermentative ammonification rather than detoxification. We conclude that fermentative ammonification by Nir allows for the energetically favorable fermentation of glucose to formate and acetate. These results and conclusions are discussed in light of the roles of Nir in other bacteria and in plants.

Nitrogen oxyanion-dependent dissociation of a two-component complex that regulates bacterial nitrate assimilation.

Nitrogen is an essential nutrient for growth and is readily available to microbes in many environments in the form of ammonium and nitrate. Both ions are of environmental significance due to sustained use of inorganic fertilizers on agricultural soils. Diverse species of bacteria that have an assimilatory nitrate/nitrite reductase system (NAS) can use nitrate or nitrite as the sole nitrogen source for growth when ammonium is limited. In Paracoccus denitrificans, the pathway-specific two-component regulator for NAS expression is encoded by the nasT and nasS genes. Here, we show that the putative RNA-binding protein NasT is a positive regulator essential for expression of the nas gene cluster (i.e. nasABGHC). By contrast, a nitrogen oxyanion-binding sensor (NasS) is required for nitrate/nitrite-responsive control of nas gene expression. The NasS and NasT proteins co-purify as a stable heterotetrameric regulatory complex, NasS-NasT. This protein-protein interaction is sensitive to nitrate and nitrite, which cause dissociation of the NasS-NasT complex into monomeric NasS and an oligomeric form of NasT. NasT has been shown to bind the leader RNA for nasA. Thus, upon liberation from the complex, the positive regulator NasT is free to up-regulate nas gene expression.

X-ray crystal structure of a mutant assimilatory nitrite reductase that shows sulfite reductase-like activity.

Assimilatory nitrite reductase (aNiR) reduces nitrite ions (NO(2)(-)) to ammonium ions (NH(4)(+)), whereas assimilatory sulfite reductase reduces sulfite (SO(3)(2-)) to hydrogen sulfide (HS(-)). Although aNiR can also reduce SO(3)(2-), its activity is much lower than when NO(2)(-) is reduced as the substrate. To increase the SO(3)(2-)-reduction activity of aNiR, we performed a N226K mutation of Nii3, a representative aNiR. The resulting Nii3-N226K variant could bind non-native targets, SO(3)(2-), and HCO(3)(-), in addition to its native target, i.e., NO(2)(-). We have determined the high-resolution structure of Nii3-N226K in its apo-state and in complex with SO(3)(2-), NO(2)(-), and HCO(3)(-). This analysis revealed conformational changes of Lys226 and the adjacent Lys224 upon binding of SO(3)(2-), but not NO(2)(-)In contrast, HCO(3)(-) binding induced a conformational change at Arg179. After replacing Asn226 with a positively charged Lys, aNiR showed affinity for several anions. A comparison of all ligand-bound structures for Nii3-N226K revealed that structural changes in the active site depend on the size of the substrate.

Eukaryotic assimilatory nitrate reductase fractionates N and O isotopes with a ratio near unity.

In order to (i) establish the biological systematics necessary to interpret nitrogen (N) and oxygen (O) isotope ratios of nitrate ((15)N/(14)N and (18)O/(16)O) in the environment and (ii) investigate the potential for isotopes to elucidate the mechanism of a key N cycle enzyme, we measured the nitrate N and O isotope effects ((15)ε and (18)ε) for nitrate reduction by two assimilatory eukaryotic nitrate reductase (eukNR) enzymes. The (15)ε for purified extracts of NADPH eukNR from the fungus Aspergillus niger and the (15)ε for NADH eukNR from cell homogenates of the marine diatom Thalassiosira weissflogii were indistinguishable, yielding a mean (15)ε for the enzyme of 26.6 ± 0.2‰. Both forms of eukNR imparted near equivalent fractionation on N and O isotopes. The increase in (18)O/(16)O versus the increase in (15)N/(14)N (relative to their natural abundances) was 0.96 ± 0.01 for NADPH eukNR and 1.09 ± 0.03 for NADH eukNR. These results are the first reliable measurements of the coupled N and O isotope effects for any form of eukNR. They support the prevailing view that intracellular reduction by eukNR is the dominant step in isotope fractionation during nitrate assimilation and that it drives the (18)ε:(15)ε ≈ 1 observed in phytoplankton cultures, suggesting that this O-to-N isotope signature will apply broadly in the environment. Our measured (15)ε and (18)ε may represent the intrinsic isotope effects for eukNR-mediated N-O bond rupture, a potential constraint on the nature of the enzyme's transition state.

Structure-function relationship of assimilatory nitrite reductases from the leaf and root of tobacco based on high-resolution structures.

Tobacco expresses four isomers of assimilatory nitrite reductase (aNiR), leaf-type (Nii1 and Nii3), and root-type (Nii2 and Nii4). The high-resolution crystal structures of Nii3 and Nii4, determined at 1.25 and 2.3 Å resolutions, respectively, revealed that both proteins had very similar structures. The Nii3 structure provided detailed geometries for the [4Fe-4S] cluster and the siroheme prosthetic groups. We have generated two types of Nii3 variants: one set focuses on residue Met175 (Nii3-M175G, Nii3-M175E, and Nii3-M175K), a residue that is located on the substrate entrance pathway; the second set targets residue Gln448 (Nii3-Q448K), a residue near the prosthetic groups. Comparison of the structures and kinetics of the Nii3 wild-type (Nii3-WT) and the Met175 variants showed that the hydrophobic side-chain of Met175 facilitated enzyme efficiency (k(cat) /K(m) ). The Nii4-WT has Lys449 at the equivalent position of Gln448 in Nii3-WT. The enzyme activity assay revealed that the turnover number (k(cat) ) and Michaelis constant (K(m) ) of Nii4-WT were lower than those of Nii3-WT. However, the k(cat) /K(m) of Nii4-WT was about 1.4 times higher than that of Nii3-WT. A comparison of the kinetics of the Nii3-Q448K and Nii4-K449Q variants revealed that the change in k(cat) /K(m) was brought about by the difference in Residue 448 (defined as Gln448 in Nii3 and Lys449 in Nii4). By combining detailed crystal structures with enzyme kinetics, we have proposed that Nii3 is the low-affinity and Nii4 is the high-affinity aNiR.

Identification and functional analysis of a nitrate assimilation operon nasACKBDEF from Amycolatopsis mediterranei U32.

Nitrate assimilation has been well studied for Gram-negative bacteria but not so much in the Gram-positive actinomycetes up to date. In a rifamycin SV-producing actinomycete, Amycolatopsis mediterranei strain U32, nitrate not only can be used as a sole nitrogen source but also remarkably stimulates the antibiotic production along with regulating the related metabolic enzymes. A gene cluster of nasACKBDEF was cloned from a U32 genomic library by in situ hybridization screening with a heterogeneous nasB probe and confirmed later by whole genome sequence, corresponding to the protein coding genes of AMED_1121 to AMED_1127. These genes were co-transcribed as an operon, concomitantly repressed by ammonium while activated with supplement of either nitrate or nitrite. Genetic and biochemical analyses identified the essential nitrate/nitrite assimilation functions of the encoded proteins, orderly, the assimilatory nitrate reductase catalytic subunit (NasA), nitrate reductase electron transfer subunit (NasC), nitrate/nitrite transporter (NasK), assimilatory nitrite reductase large subunit (NasB) and small subunit (NasD), bifunctional uroporphyrinogen-III synthase (NasE), and an unknown function protein (NasF). Comparing rifamycin SV production and the level of transcription of nasB and rifE from U32 and its individual nas mutants in Bennet medium with or without nitrate indicated that nitrate assimilation function encoded by the nas operon played an essential role in the "nitrate stimulated" rifamycin production but had no effect upon the transcription regulation of the primary and secondary metabolic genes related to rifamycin biosynthesis.

The effect of ammonium on assimilatory nitrate reduction in the haloarchaeon Haloferax mediterranei.

Physiology, regulation and biochemical aspects of the nitrogen assimilation are well known in Prokarya or Eukarya but they are poorly described in Archaea domain. The haloarchaeon Haloferax mediterranei can use different nitrogen inorganic sources (NO (3) (-) , NO (2) (-) or NH (4) (+) ) for growth. Different approaches were considered to study the effect of NH (4) (+) on nitrogen assimilation in Hfx. mediterranei cells grown in KNO(3) medium. The NH (4) (+) addition to KNO(3) medium caused a decrease of assimilatory nitrate (Nas) and nitrite reductases (NiR) activities. Similar effects were observed when nitrate-growing cells were transferred to NH (4) (+) media. Both activities increased when NH (4) (+) was removed from culture, showing that the negative effect of NH (4) (+) on this pathway is reversible. These results suggest that ammonium causes the inhibition of the assimilatory nitrate pathway, while nitrate exerts a positive effect. This pattern has been confirmed by RT-PCR. In the presence of both NO (3) (-) and NH (4) (+) , NH (4) (+) was preferentially consumed, but NO (3) (-) uptake was not completely inhibited by NH (4) (+) at prolonged time scale. The addition of MSX to NH (4) (+) or NO (3) (-) cultures results in an increase of Nas and NiR activities, suggesting that NH (4) (+) assimilation, rather than NH (4) (+ ) per se, has a negative effect on assimilatory nitrate reduction in Hfx. mediterranei.