Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species.
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We examined the ability of plant nitrate reductase (NR) to produce nitric oxide (NO) using in vitro assays. Electrochemical and fluorometric measurements both showed that NO is produced by corn NR in the presence of nitrite and NADH at pH 7. The NO production was inhibited by sodium azide, a known inhibitor for NR. During the reaction, absorbance of 2',7'-dichlorodihydrofluorescein increased markedly. This change was completely suppressed by sodium azide, glutathione or depletion of oxygen. We conclude that plant NR produces both NO and its toxic derivative, peroxynitrite, under aerobic conditions when nitrite is provided as the substrate for NR. (+info)
Nitrate assimilation genes of the marine diazotrophic, filamentous cyanobacterium Trichodesmium sp. strain WH9601.
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A 4.0-kb DNA fragment of Trichodesmium sp. strain WH9601 contained gene sequences encoding the nitrate reduction enzymes, nirA and narB. A third gene positioned between nirA and narB encodes a putative membrane protein with similarity to the nitrate permeases of Bacillus subtilis (NasA) and Emericella nidulans (CrnA). The gene was shown to functionally complement a DeltanasA mutant of B. subtilis and was assigned the name napA (nitrate permease). NapA was involved in both nitrate and nitrite uptake by the complemented B. subtilis cells. napA is distinct from the nrt genes that encode the nitrate transporter of freshwater cyanobacteria. (+info)
Purification and characterization of dissimilatory nitrate reductase from a denitrifying halophilic archaeon, Haloarcula marismortui.
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Dissimilatory nitrate reductase was purified from a denitrifying halophilic archaeon, Haloarcula marismortui, to an electrophoretically homogeneous state. The purified enzyme was inferred to be a homotetramer composed of a 63 kDa polypeptide. The electron paramagnetic resonance spectrum of the purified enzyme revealed typical rhombic signals which were ascribed to Mo(V) in the Mo-molybdopterin complex. Like the bacterial membrane-bound (Nar-) enzyme, the purified enzyme supported the catalysis of chlorate. The enzyme was activated in extreme saline conditions and the values of k(cat) and K(m) toward nitrate were 145 s(-1) and 79 microM, respectively, in the presence of 2.0 M NaCl. (+info)
Two nitrate/nitrite transporters are encoded within the mobilizable plasmid for nitrate respiration of Thermus thermophilus HB8.
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Thermus thermophilus HB8 can grow anaerobically by using a membrane-bound nitrate reductase to catalyze the reduction of nitrate as a final electron acceptor in respiration. In contrast to other denitrifiers, the nitrite produced does not continue the reduction pathway but accumulates in the growth medium after its active extrusion from the cell. We describe the presence of two genes, narK1 and narK2, downstream of the nitrate reductase-encoding gene cluster (nar) that code for two homologues to the major facilitator superfamily of transporters. The sequences of NarK1 and NarK2 are 30% identical to each other, but whereas NarK1 clusters in an average-distance tree with putative nitrate transporters, NarK2 does so with putative nitrite exporters. To analyze whether this differential clustering was actually related to functional differences, we isolated derivatives with mutations of one or both genes. Analysis revealed that single mutations had minor effects on growth by nitrate respiration, whereas a double narK1 narK2 mutation abolished this capability. Further analysis allowed us to confirm that the double mutant is completely unable to excrete nitrite, while single mutants have a limitation in the excretion rates compared with the wild type. These data allow us to propose that both proteins are implicated in the transport of nitrate and nitrite, probably acting as nitrate/nitrite antiporters. The possible differential roles of these proteins in vivo are discussed. (+info)
Structure-function analysis of NADPH:nitrate reductase from Aspergillus nidulans: analysis of altered pyridine nucleotide specificity in vivo.
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Nitrate reductase (NaR) catalyses the reduction of nitrate to nitrite via a two-electron transfer. In fungi, the electron donor for NaR is NADPH whereas plants can have two enzymes, NADH:NaR and a bispecific NAD(P)H:NaR. PCR mutagenesis was employed to introduce mutations into the niaD gene of Aspergillus nidulans in order to identify residues involved in co-enzyme specificity. The niaD3000 mutation (NiaD T813D, K814Q) altered co-enzyme specificity: the new enzyme had high levels of NADH:NaR activity in vitro, whilst all NADPH-associated activity was lost. However, strains carrying this mutation did not grow on nitrate. Enzyme assays suggested that this was not due to inhibition of the mutant enzyme by NADPH. All revertants of the niaD3000 mutants had restored NADPH activity and lost NADH activity. Sequence analysis of these revertants showed that they all contained a single amino acid change at Asp-813, suggesting that this position is crucial to co-enzyme specificity. Further studies have shown that the mutant enzyme was not protected from deactivation by either co-factor in cell-free extracts (unlike the wild-type), and that induction of the glucose-6-phosphate dehydrogenase occurred independently of NADPH levels. These data highlight the importance of functional tests in vivo under physiological conditions. (+info)
Inactivation of gltB abolishes expression of the assimilatory nitrate reductase gene (nasB) in Pseudomonas putida KT2442.
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By using mini-Tn5 transposon mutagenesis, random transcriptional fusions of promoterless bacterial luciferase, luxAB, to genes of Pseudomonas putida KT2442 were generated. Insertion mutants that responded to ammonium deficiency by induction of bioluminescence were selected. The mutant that responded most strongly was genetically analyzed and is demonstrated to bear the transposon within the assimilatory nitrate reductase gene (nasB) of P. putida KT2442. Genetic evidence as well as sequence analyses of the DNA regions flanking nasB suggest that the genes required for nitrate assimilation are not clustered. We isolated three second-site mutants in which induction of nasB expression was completely abolished under nitrogen-limiting conditions. Nucleotide sequence analysis of the chromosomal junctions revealed that in all three mutants the secondary transposon had inserted at different sites in the gltB gene of P. putida KT2442 encoding the major subunit of the glutamate synthase. A detailed physiological characterization of the gltB mutants revealed that they are unable to utilize a number of potential nitrogen sources, are defective in the ability to express nitrogen starvation proteins, display an aberrant cell morphology under nitrogen-limiting conditions, and are impaired in the capacity to survive prolonged nitrogen starvation periods. (+info)
Recombinant expression of molybdenum reductase fragments of plant nitrate reductase at high levels in Pichia pastoris.
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Mo reductase (MoR; formerly cytochrome c reductase) fragments of NADH:NO(3) reductase (NR; EC1.6.6.1) were cytosolically expressed in Pichia pastoris, a methylotrophic yeast, using spinach (Spinacia oleracea) and corn (Zea maize) cDNAs. In fermenter cultures, spinach MoR was expressed at 420 mg L(-1), corn MoR at 32 mg L(-1), and corn MoR plus with putative NR interface domain N terminus (MoR+) at 17 mg L(-1). Constitutively expressed MoR+ was structurally stable while it was degraded when expressed by methanol induction, which suggests methanol growth produces more proteinase. Methanol-induced expression yielded more target protein. All three MoR were purified to homogeneity and their polypeptides were approximately 41 (MoR) and approximately 66 (MoR+) kD. MoR was monomeric and MoR+ dimeric, confirming the predicted role for dimer interface domain of NR. MoR+, although differing in quaternary structure from MoR, has similar kinetic properties for ferricyanide and cytochrome c reductase activities and visible spectra, which were like NR. Redox potentials of MoR and MoR+ were similar for flavin, whereas MoR+ had a more negative potential for heme-iron. Reaction schemes for MoR catalyzed reactions were proposed based on fast-reaction rapid-scan stopped-flow kinetic analysis of MoR. P. pastoris is an excellent system for producing the large amounts of NR fragments needed for detailed biochemical studies. (+info)
Deletion of the nitrate reductase N-terminal domain still allows binding of 14-3-3 proteins but affects their inhibitory properties.
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Nitrate reductase (NR) is post-translationally regulated by phosphorylation and binding of 14-3-3 proteins. Deletion of 56 amino acids in the amino-terminal domain of NR was previously shown to impair this type of regulation in tobacco (Nicotiana plumbaginifolia) (L. Nussaume, M. Vincentez, C. Meyer, J.-P. Boutin, M. Caboche [1995] Plant Cell 7: 611-621), although both full-length NR and deleted NR (DeltaNR) appeared to be phosphorylated in darkness (C. Lillo, S. Kazazaic, P. Ruoff, C. Meyer [1997] Plant Physiol 114: 1377-1383). We show here that in the presence of Mg(2+) and phosphatase inhibitors, NR and endogenous 14-3-3 proteins copurify through affinity chromatography. Assay of NR activity and western blots showed that endogenous 14-3-3 proteins copurified with both NR and DeltaNR. Electron transport in the heme-binding domain of DeltaNR was inhibited by Mg(2+)/14-3-3, whereas this was not the case for NR. This may indicate a different way of binding for 14-3-3 in the DeltaNR compared with NR. The DeltaNR was more labile than NR, in vitro. Lability was ascribed to the molybdopterin binding domain, and apparently an important function of the 56 amino acids is stabilization of this domain. (+info)