15N Tracing Studies on In Vitro Reactions of Ferredoxin-Dependent Nitrite Reductase and Glutamate Synthase Using Reconstituted Electron Donation Systems.

It is known that plants contain ferredoxin (Fd)-dependent nitrite reductase (NiR) and glutamate synthase (GOGAT). The Fd-NiR reaction produces ammonia from nitrite, and the activity is usually measured by nitrite disappearance. The Fd-GOGAT reaction forms two glutamates of different origin, from glutamine and 2-oxoglutarate, and the activity is measured by the oxidation of reductant (NADPH) or by formation of total glutamate. Here, a quantitative probe of the products and efficiency of the process was conducted using (15)N tracing techniques on these reactions in vitro. We quantified the reduction of (15)N-labeled [Formula: see text] to [Formula: see text] and the formation of [(15)N]glutamate and [(14)N]glutamate from [5-(15)N-amide]glutamine plus 2-oxoglutarate by NiR and GOGAT, respectively, with the reductant-Fd-NADP(+) oxidoreductase (FNR)-Fd system as the sequential electron donors. The supply of dithionite or NADPH to recombinant cyanobacterial NiR led to electron donation system-dependent formation of [(15)N]ammonium from [(15)N]nitrite. Addition of 20 mM NaCl and 20 mM Na-ascorbate accelerated nitrite reduction under high concentrations of NADPH. A sufficient supply of NADPH to recombinant Zea mays Fd-GOGAT generated complete GOGAT activity (transferring the [5-(15)N]amide of glutamine to 2-oxoglutarate to form [(15)N]glutamate), whereas a shortage of NADPH resulted in glutaminase activity only, which removed the amide from glutamine and released ammonia and [(14)N]glutamate. We conclude that although the recombinant Fd-GOGAT enzyme has two forms of glutamate synthesis, the first by glutaminase (ammonia release by glutamine amidotransferase) and the second by glutamate synthase (coupling of the ammonia and exogenously applied 2-oxoglutarate), the first works without NADPH, while the second is strictly dependent on NADPH availability.

Agrobacterium-mediated co-transformation of rice using two selectable marker genes derived from rice genome components.

A method for Agrobacterium-mediated co-transformation of rice (Oryza sativa L.) was developed using rice-derived selection markers. Two T-DNAs were efficiently introduced into separate loci using selectable marker gene cassettes consisting of the mutated acetolactate synthase gene (mALS) under the control of the callus-specific promoter (CSP) (CSP:mALS) and the ferredoxin nitrite reductase gene (NiR) under the control of its own promoter (NiR P:NiR). The CSP:mALS gene cassette confers sulfonylurea herbicide resistance to transgenic rice callus. The NiR P:NiR construct complements NiR-deficient mutant cultivars such as 'Koshihikari', which are defective in the regulation of nitrogen metabolism. In the present study, the CaMV35S:GUS and CaMV35S:GFP gene cassettes were co-introduced into the 'Koshihikari' genome using our system. Approximately 5-10 independent transgenic lines expressing both the GUS and GFP reporters were obtained from 100 Agrobacterium co-inoculated calli. Furthermore, transgenic 'Koshihikari' rice lines with reduced content of two major seed allergen proteins, the 33 and 14-16 kDa allergens, were generated by this co-transformation system. The present results indicate that the generation of selectable antibiotic resistance marker gene-free transgenic rice is possible using our rice-derived selection marker co-transformation system. Key message An improved rice transformation method was developed based on Agrobacterium-mediated co-transformation using two rice genome-derived selectable marker gene cassettes.

Nitroreductase activity of ferredoxin reductase BphA4 from Dyella ginsengisoli LA-4 by catalytic and structural properties analysis.

Ferredoxin reductase BphA4 was well known as a component of biphenyl dioxygenase. However, there was little information about whether it could utilize nonphysiological oxidants as electron acceptors. In the present study, we reported the novel nitroreductase activity of BphA4(LA)₋4. The homology model of ferredoxin reductase BphA4 from Dyella ginsengisoli LA-4 was constructed. According to the alignment of three-dimensional structures, it was supposed that BphA4(LA)₋4 could function as nitroreductase. Recombinant His-tagged BphA4(LA)₋4 was purified with a molecular mass of 49.6 ± 1 kDa. Biochemical characterization of purified BphA4(LA)₋4 possessed the nitroreductase activity with the optimal temperature 50°C and pH 8.0. The substrate spectrum and kinetics indicated BphA4(LA)₋4 could reduce several nitroaromatics with different apparent K(m) values: m-dinitrobenzene (560 µM), o-dinitrobenzene (1,060 µM), o-nitroaniline (1,570 µM), m-nitrobenzoic acid (1,300 µM) and m-nitrophenol (67 µM). The nitroreductase activity was further explained by docking studies, which was indicated that Arg 288 should play an important role in binding nitroaromatics. Moreover, there existed a good linear correlation between lnK(m) and calculated binding energy.

Consumption of hydrogen ions in rapid-equilibrium enzyme kinetics.

Enzyme-catalyzed reductase reactions in particular are characterized by large changes in the binding of hydrogen ions Δ(r)N(H). This is a thermodynamic property of the reaction that is catalyzed. For example, in the ferredoxin-nitrite reductase reaction, there is an increase of eight in the binding of hydrogen ions for every molecule of nitrite reduced to ammonia H(2)O. If these hydrogen ions are consumed in the rate-determining reaction, the limiting velocity is proportional to [H(+)](8). This would make it practically impossible to determine the kinetic parameters. This article shows that when n hydrogen ions are consumed in reactions preceding the rate-determining reaction the limiting velocity is not proportional to [H(+)](n) and may only vary with pH according to the pK's of the enzyme-substrate complex that produces products. Rapid-equilibrium rate equations for ordered A + B → products are derived for two mechanisms in which a single hydrogen ion is consumed prior to the rate-determining reaction. Rate equations are tested by calculating velocities for the minimum number of velocity measurements required to estimate the kinetic parameters and using these velocities to estimate the kinetic parameters.

Structural enzymology of sulphur metabolism in Mycobacterium tuberculosis.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis poses a serious threat to human health and has led to world-wide efforts focusing on the development of novel vaccines and antibiotics against this pathogen. Sulphur metabolism in this organism has been linked to essential processes such as virulence and redox defence. The cysteine biosynthetic pathway is up-regulated in models of persistent M. tuberculosis infections and provides potential targets for novel anti-mycobacterial agents, directed specifically toward the pathogen in its persistent phase. Functional and structural characterization of enzymes from sulfur metabolism establishes a necessary framework for the design of strong binding inhibitors that might be developed into new drugs. This review summarizes recent progress in the elucidation of the structural enzymology of the sulphate reduction and cysteine biosynthesis pathways.

Enzymatic properties of the ferredoxin-dependent nitrite reductase from Chlamydomonas reinhardtii. Evidence for hydroxylamine as a late intermediate in ammonia production.

The ferredoxin-dependent nitrite reductase from the green alga Chlamydomonas reinhardtii has been cloned, expressed in Escherichia coli as a His-tagged recombinant protein, and purified to homogeneity. The spectra, kinetic properties and substrate-binding parameters of the C. reinhardtii enzyme are quite similar to those of the ferredoxin-dependent spinach chloroplast nitrite reductase. Computer modeling, based on the published structure of spinach nitrite reductase, predicts that the structure of C. reinhardtii nitrite reductase will be similar to that of the spinach enzyme. Chemical modification studies and the ionic-strength dependence of the enzyme's ability to interact with ferredoxin are consistent with the involvement of arginine and lysine residues on C. reinhardtii nitrite reductase in electrostatically-stabilized binding to ferredoxin. The C. reinhardtii enzyme has been used to demonstrate that hydroxylamine can serve as an electron-accepting substrate for the enzyme and that the product of hydroxylamine reduction is ammonia, providing the first experimental evidence for the hypothesis that hydroxylamine, bound to the enzyme, can serve as a late intermediate during the reduction of nitrite to ammonia catalyzed by the enzyme.

The interaction of spinach nitrite reductase with ferredoxin: a site-directed mutation study.

A series of site-directed mutants of the ferredoxin-dependent spinach nitrite reductase has been characterized and several amino acids have been identified that appear to be involved in the interaction of the enzyme with ferredoxin. In a complementary study, binding constants to nitrite reductase and steady-state kinetic parameters of site-directed mutants of ferredoxin were determined in an attempt to identify ferredoxin amino acids involved in the interaction with nitrite reductase. The results have been interpreted in terms of an in-silico docking model for the 1:1 complex of ferredoxin with nitrite reductase.

Fhit interaction with ferredoxin reductase triggers generation of reactive oxygen species and apoptosis of cancer cells.

Fhit protein is lost in most cancers, its restoration suppresses tumorigenicity, and virus-mediated FHIT gene therapy induces apoptosis and suppresses tumors in preclinical models. We have used protein cross-linking and proteomics methods to characterize a Fhit protein complex involved in triggering Fhit-mediated apoptosis. The complex includes Hsp60 and Hsp10 that mediate Fhit stability and may affect import into mitochondria, where it interacts with ferredoxin reductase, responsible for transferring electrons from NADPH to cytochrome P450 via ferredoxin. Viral-mediated Fhit restoration increases production of intracellular reactive oxygen species, followed by increased apoptosis of lung cancer cells under oxidative stress conditions; conversely, Fhit-negative cells escape apoptosis, carrying serious oxidative DNA damage that may contribute to an increased mutation rate. Characterization of Fhit interacting proteins has identified direct effectors of the Fhit-mediated apoptotic pathway that is lost in most cancers through loss of Fhit.

Three mechanisms and rapid-equilibrium rate equations for a type of reductase reaction.

Rapid-equilibrium rate equations for enzyme-catalyzed reactions are especially useful when the mechanism involves a number of pKs, but they are also useful when some reactants have stoichiometric numbers greater than one or hydrogen ions are produced or consumed in the rate-determining step. The pH dependencies of limiting velocities, Michaelis constants, and reaction velocities for the forward reaction are discussed for two examples of reductase reactions of the type mR + O -> products, where R is the reductant and O is the oxidant. For the nitrate reductase reaction (EC 1.9.6.1), m = 2 and two hydrogen ions are consumed. For the nitrite-ferredoxin reductase reaction (EC 1.7.7.1), m = 6 and eight hydrogen ions are consumed. The expressions for the limiting velocities, Michaelis constants, and rate equations for the forward reaction are derived for two ordered mechanisms and the random mechanism. Three Mathematica programs are used to make plots of kinetic parameters as functions of pH and three-dimensional plots of rapid-equilibrium velocities as functions of [O] and [R] for arbitrary sets of input parameters.

Orthric Rieske dioxygenases for degrading mixtures of 2,4-dinitrotoluene/naphthalene and 2-amino-4,6-dinitrotoluene/4-amino-2,6-dinitrotoluene.

Pollutants are frequently found as mixtures yet it is difficult to engineer enzymes with broad substrate ranges on aromatics. Inspired by the archetypal nitroarene dioxygenase, which shares its electron transport with a salicylate monooxygenase, we have created an innovative and general approach to expand the substrate range of dioxygenase enzymes in a single cell. We have developed here a series of novel, hybrid dioxygenase enzymes that function with a single ferredoxin reductase and ferredoxin that are used to transport two electrons from nicotinamide adenine dinucleotide to the two independent terminal oxygenases. Each independent alpha-oxygenase may then be used simultaneously to create orthric enzymes that degrade mixtures of environmental pollutants. Specifically, we created a hybrid dioxygenase system consisting of naphthalene dioxygenase/dinitrotoluene dioxygenase to simultaneously degrade 2,4-dinitrotoluene and naphthalene (neither enzyme alone had significant activity on both compounds) and dinitrotoluene dioxygenase/nitrobenzene dioxygenase to simultaneously degrade the frequently encountered 2,4,6-trinitrotoluene reduction products 2-amino-4,6-dinitrotoluene and 4-amino-2,6-dinitrotoluene.

Identification and transcriptional analysis of nitrate assimilation genes in the halophilic archaeon Haloferax mediterranei.

Sequencing a 6,720-bp segment of the extreme halophilic archaeon Haloferax mediterranei genome has revealed the genomic organization of the putative structural genes for nitrate assimilation. We report a gene operon containing nasABC and nasD gene. nasA encodes an assimilatory nitrate reductase, nasB codes for a membrane protein with similarity to the NarK transporter, nasC encodes a protein with similarity to MobA; and nasD codes for an assimilatory ferredoxin-dependent nitrite reductase. Reverse transcription-PCR and primer extension experiments have demonstrated the existence of one polycistronic messenger nasABC and one monocistronic nasD initiated from a different promoter. The gene order and the grouping in two adjacent transcriptional units constitutes a novel organization of nas genes. The promoter regions harbor direct palindromes reminiscent of target sites for binding of a hypotetical regulatory protein(s). Transcription of the nasABC and nasD regions was found to be repressed by the presence of ammonium as nitrogen source.

Isolation of a rice regeneration quantitative trait loci gene and its application to transformation systems.

Regeneration of plant organs is often the essential step in genetic transformation; however, the regeneration ability of a plant varies depending on the genetic background. By conventional crosses of low-regeneration rice strain Koshihikari with high-regeneration rice strain Kasalath, we identified some quantitative trait loci, which control the regeneration ability in rice. Using a map-based cloning strategy, we isolated a main quantitative trait loci gene encoding ferredoxin-nitrite reductase (NiR) that determines regeneration ability in rice. Molecular analyses revealed that the poor regeneration ability of Koshihikari is caused by lower expression than in Kasalath and the specific activity of NiR. Using the NiR gene as a selection marker, we succeeded in selectively transforming a foreign gene into rice without exogenous marker genes. Our results demonstrate that nitrate assimilation is an important process in rice regeneration and also provide an additional selectable marker for rice transformation.

Nitrite accumulation and nitric oxide emission in relation to cellular signaling in nitrite reductase antisense tobacco.

An antisense nitrite reductase (NiR, EC 1.7.7.1) tobacco ( Nicotiana tabacum L.) transformant (clone 271) was used to gain insight into a possible correlation between nitrate reductase (NR, EC 1.6.6.1)-dependent nitrite accumulation and nitric oxide (NO(.)) production, and to assess the regulation of signal transduction in response to stress conditions. Nitrite concentrations of clone 271 leaves were 10-fold, and NO(.) emission rates were 100-fold higher than in wild type leaves. Increased protein tyrosine nitration in clone 271 suggests that high NO(.) production resulted in increased peroxynitrite (ONOO(-)) formation. Tyrosine nitration was also observed in vitro by adding peroxynitrite to leaf extracts. As in mammalian cells, NO(.) and derivatives also increased synthesis of proteins like 14-3-3 and cyclophilins, which are both involved in regulation of activity and stability of enzymes.

The ferredoxin-binding site of ferredoxin: Nitrite oxidoreductase. Differential chemical modification of the free enzyme and its complex with ferredoxin.

Spinach (Spinacea oleracea) leaf ferredoxin (Fd)-dependent nitrite reductase was treated with either the arginine-modifying reagent phenyl-glyoxal or the lysine-modifying reagent pyridoxal-5'-phosphate under conditions where only the Fd-binding affinity of the enzyme was affected and where complex formation between Fd and the enzyme prevented the inhibition by either reagent. Modification with [14C]phenylglyoxal allowed the identification of two nitrite reductase arginines, R375 and R556, that are protected by Fd against labeling. Modification of nitrite reductase with pyridoxal-5'-phosphate, followed by reduction with NaBH4, allowed the identification of a lysine, K436, that is protected by Fd against labeling. Positive charges are present at these positions in all of the Fd-dependent nitrite reductase for which sequences are available, suggesting that these amino acids are directly involved in electrostatic binding of Fd to the enzyme.

Nitrogen deprivation of Anabaena sp. strain PCC 7120 elicits rapid activation of a gene cluster that is essential for uptake and utilization of nitrate.

A transposon bearing luxAB, encoding luciferase, as a reporter of transcription was used to identify genes that are activated rapidly upon deprivation of Anabaena sp. strain PCC 7120 of fixed nitrogen. The three transposon-marked loci that were identified as responding most rapidly and strongly are closely linked and situated within nirA and nrtC and between nrtD and narB, genes whose products are responsible for uptake and reduction of NO2- and NO3-. A strain bearing a transcriptional fusion of narB to luxAB was constructed. Luminescence catalyzed by LuxAB was used to report on the expression of the interrupted genes. Whether these genes are regulated only coordinately is discussed.

A novel nitrite reductase gene from the cyanobacterium Plectonema boryanum.

The gene (nirA) for nitrite reductase was cloned from the nonheterocystous, filamentous cyanobacterium Plectonema boryanum. The predicted protein consists of 654 amino acids and has a calculated molecular weight of 72,135. The deduced amino acid sequence from positions 1 to 511 is strongly similar to the entire sequence of the ferredoxin-dependent nitrite reductases from other phototrophs, while the remainder of the protein is unique to the Plectonema nitrite reductase. The C-terminal portion of the protein (amino acids 584 to 654) is 30 to 35% identical to [2Fe-2S] ferredoxins from higher plants and cyanobacteria, with all of the four Cys residues involved in binding of the [2Fe-2S] cluster in the ferredoxins being conserved. Immunoblotting analysis of the extracts of P. boryanum cells showed that the NirA polypeptide has an apparent molecular mass of 75 kDa. An insertional mutant of nirA lacked the 75-kDa polypeptide, had no nitrite reductase activity, and failed to grow on nitrate and nitrite, indicating that the novel nirA is the sole nitrite reductase gene in P. boryanum and that the NirA polypeptide with the ferredoxin-like domain is the apoprotein of the functional nitrite reductase. As in Synechococcus sp. strain PCC7942, nirA is the first gene of a large transcription unit (> 7 kb in size) and is repressed by ammonium and derepressed simply by deprivation of ammonium from the medium. The development of nitrite reductase activity was, however, found to require the presence of nitrate in the medium.

Cloning and nucleotide sequence of a leaf ferredoxin-nitrite reductase cDNA of rice.

A ferredoxin-nitrite reductase (EC 1.7.7.1) cDNA was isolated and sequenced from a lambda gt 11 cDNA library constructed from nitrate-induced greening shoots of rice (Oryza sativa L.) seedlings. The nucleotide sequence of the cDNA clone contains an open reading frame of 1788 nucleotides. There exists a strong bias for the third codon usage of G/C (95.5%) as in the case of the maize enzyme. The deduced amino acid sequence shows an overall homology to the maize (81%) and the dicot enzymes (70-74%), suggesting that the primary structure of ferredoxin-nitrite reductase is highly conserved in higher plants.

Regulation of the expression of ferredoxin-nitrite reductase in synchronous cultures of Chlamydomonas reinhardtii.

The regulation of ferredoxin-nitrite reductase--the second enzyme involved in the nitrate assimilatory pathway--in synchronous cultures of C. reinhardtii has been studied both at the activity and protein levels using specific antibodies. During a cycle of 12 h light/12 h dark (12L:12D), ferredoxin-nitrite reductase activity shows a 24-h fluctuation with a maximum in the middle of the light period. The increase of activity during the first few hours of the light phase is due to de novo synthesis of the enzyme. This synthesis occurs in the absence of NH4+ and it is highly induced by either nitrate or nitrite, but it does not require light so long as carbon skeletons are available. The decrease of ferredoxin-nitrite reductase activity during the last hours of the light period and during the dark phase is suggested to be due to protein degradation, although this process is slow because of the high stability of the enzyme. The changes in the level of ferredoxin-nitrite reductase seem to be related to events in the cell cycle under the illumination conditions used. Thus, synthesis of the enzyme correlates to growth periods within the cell cycle, and it does not seem to be under the control of a circadian rhythm.