Assimilatory nitrate reductase from the haloarchaeon Haloferax mediterranei: purification and characterisation

Haloferax mediterranei can use nitrate as sole nitrogen source during aerobic growth. We report here the purification and biochemical characterisation of the assimilatory nitrate reductase (EC 1.6.6.2) from H. mediterranei. The enzyme, as isolated, was composed of two subunits (105+/-1.3 kDa and 50+/-1.3 kDa) and behaved as a dimer during gel filtration (132+/-6 kDa). A pH of 9 and elevated temperatures up to 80 degrees C (at 3.1 M NaCl) are necessary for optimum activity. The enzyme stability and activity of the enzyme depend upon the salt concentration. Reduced methyl viologen was as effective as the natural electron donor ferredoxin in the catalytic process. In contrast, NADPH and NADH, which are electron donors in nitrate reductases from different non-photosynthetic bacteria, were ineffective.     

Conformational change of Arabidopsis thaliana thioredoxin reductase after binding of pyridine nucleotide and thioredoxin

We have found that the binding of NADP+ (Kd = 0.86+/-0.11 microM) enhanced the FAD fluorescence of Arabidopsis thaliana NADPH:thioredoxin reductase (TR, EC 1.6.4.5) by 2 times, whereas the binding of 3-aminopyridine adenine dinucleotide phosphate (AADP+) (Kd < 0.1 microM) quenched the fluorescence by 20%. Thioredoxin (TRX) also enhanced the FAD fluorescence by 35%. The Kd of TR-NADP+ and TR-AADP+ complexes did not change in the presence of 45 microM TRX. Our findings imply that the binding of NADP+ and AADP+ at the NADP(H)-binding site of A. thaliana TR, and/or the binding of TRX in the vicinity of the catalytic disulfide increase the content of fluorescent FR conformer (NADP(H)-binding site adjacent to flavin). The different effects of NADP+ and AADP+ on FAD fluorescence intensity may be explained by the superposition of two opposite factors: i) increased content of fluorescent FR conformer upon binding of NADP+ or AADP+; ii) quenching of FAD fluorescence by electron-donating 3-aminopyridinium ring of AADP+.     

Deconstructing the electron transfer chain in a complex molybdoenzyme: Assimilatory nitrate reductase from Neurospora crassa

Nitrate reductase (NR) from the fungus Neurospora crassa is a complex homodimeric metallo-flavoenzyme, where each protomer contains three distinct domains; the catalytically active terminal molybdopterin cofactor, a central heme-containing domain, and an FAD domain which binds with the natural electron donor NADPH. Here, we demonstrate the catalytic voltammetry of variants of N. crassa NRs on a modified Au electrode with the electrochemically reduced forms of benzyl viologen (BV²⁺) and anthraquinone sulfonate (AQS^?) acting as artificial electron donors. The biopolymer chitosan used to entrap NR on the electrode non-covalently and the enzyme film was both stable and highly active. Electrochemistry was conducted on two distinct forms; one lacking the FAD cofactor and the other lacking both the FAD and heme cofactors. While both enzymes showed catalytic nitrate reductase activity, removal of the heme cofactor resulted in a more significant effect on the rate of nitrate reduction. Electrochemical simulation was carried out to enable kinetic characterisation of both the NR:nitrate and NR:mediator reactions.     

Optimization of binding electrostatics: charge complementarity in the barnase-barstar protein complex

Theoretical and experimental studies have shown that the large desolvation penalty required for polar and charged groups frequently precludes their involvement in electrostatic interactions that contribute strongly to net stability in the folding or binding of proteins in aqueous solution near room temperature. We have previously developed a theoretical framework for computing optimized electrostatic interactions and illustrated use of the algorithm with simplified geometries. Given a receptor and model assumptions, the method computes the ligand-charge distribution that provides the most favorable balance of desolvation and interaction effects on binding. In this paper the method has been extended to treat complexes using actual molecular shapes. The barnase-barstar protein complex was investigated with barnase treated as a target receptor. The atomic point charges of barstar were varied to optimize the electrostatic binding free energy. Barnase and natural barstar form a tight complex (K(d) approximately 10(-14) M) with many charged and polar groups near the interface that make this a particularly relevant system for investigating the role of electrostatic effects on binding. The results show that sets of barstar charges (resulting from optimization with different constraints) can be found that give rise to relatively large predicted improvements in electrostatic binding free energy. Principles for enhancing the effect of electrostatic interactions in molecular binding in aqueous environments are discussed in light of the optima. Our findings suggest that, in general, the enhancements in electrostatic binding free energy resulting from modification of polar and charged groups can be substantial. Moreover, a recently proposed definition of electrostatic complementarity is shown to be a useful tool for examining binding interfaces. Finally, calculational results suggest that wild-type barstar is closer to being affinity optimized than is barnase for their mutual binding, consistent with the known roles of these proteins.     

Hysteretic behavior of nitrate reductase. Evidence of an allosteric binding site for reduced pyridine nucleotides.

In the absence of NADH, at 25 degrees C, partially purified NADH:nitrate reductase undergoes an approximately 50% reduction of its initial activity during 2 h. With the increase of inactivation, the NADH and nitrite concentration time curves become typical "sigmoidal," i.e. the reaction velocity of the nitrate reductase catalyzed reaction goes through a maximum before equilibrium is reached. About 80% of the original activity of nitrate reductase is restored when the enzyme is incubated for 2 min with 200 microM NADH or NADPH. Also other NADH substrate analogues have similar effects in restoring the lost activity. After incubation with the reduced pyridine nucleotides, the sigmoidal appearance of the NADH concentration time curve disappears almost completely. Despite the fact that NADPH increases the activity of the enzyme, NADPH does not show any competition with the NADH-binding site of nitrate reductase and does not produce nitrite in the absence of NADH. It is therefore concluded that there must be an additional allosteric site which binds either NADH or NADPH, or other pyridine nucleotides with the effect of increasing the activity of the enzyme. A kinetic model is presented which simulates the observed experimental findings.     

Bovine lens aldose reductase: tight binding of the pyridine coenzyme

Analysis by HPLC of the protein-free supernatant obtained after denaturation of aldose reductase shows that the native form of the enzyme (ARb) contains a tightly bound NADP+, which is absent in the oxidatively modified form (ARa). The absorption, fluorescence, and circular dichroism spectra of ARb and ARa are consistent with the presence of the cofactor only in the native form of aldose reductase. On the other hand, the modified enzyme, in appropriate thiol reducing conditions, can tightly bind NADP+. This indicates a potential reversibility of the modification of aldose reductase, at least in terms of retention of the cofactor.     

Relationship between pyridine nucleotide levels and ribonucleotide reductase activity in yoshida ascites hepatoma AH 130

We measured both pyridine nucleotide levels and ribonucleotide reductase-specific activity in Yoshida ascites hepatoma cells as a function of growth in vivo and during recruitment from non-cycling to cycling state in vitro. Oxidized nicotinamide adenine dinucleotide (NAD⁺) and reduced nicotinamide adenine dinucleotide (NADP) levels remained unchanged during tumour growth, while NADP⁺ and reduced nicotinamide adenine dinucleotide phosphate (NADPH) levels were very high in exponentially growing cells and markedly decreased in the resting phase. Ribonucleotide reductase activity paralleled NADP(H) (NADP⁺ plus NADPH) intracellular content. The concomitant increase in both NADP(H) levels and ribonucleotide reductase activity was also observed during G1-S transition in vitro. Cells treated with hydroxyurea showed a comparable correlation between the pool size of NADP(H) and ribonucleotide reductase activity. On the basis of these findings, we suggest that fluctuations in NADP(H) levels and ribonucleotide reductase activity might play a critical role in cell cycle regulation.     

The sequence of squash NADH:nitrate reductase and its relationship to the sequences of other flavoprotein oxidoreductases. A family of flavoprotein pyridine nucleotide cytochrome reductases.

Nucleotide sequences were determined for cDNA clones for squash NADH:nitrate oxidoreductase (EC 1.6.6.1), which is one of the most completely characterized forms of this higher plant enzyme. An open reading frame of 2754 nucleotides began at the first ATG. The deduced amino acid sequence contains 918 residues, with a predicted Mr = 103,376. The amino acid sequence is very similar to sequences deduced for other higher plant nitrate reductases. The squash sequence has significant similarity to the amino acid sequences of sulfite oxidase, cytochrome b5, and NADH:cytochrome b5 reductase. Alignment of these sequences with that of squash defines domains of nitrate reductase that appear to bind its 3 prosthetic groups (molybdopterin, heme-iron, and FAD). The amino acid sequence of the FAD domain of squash nitrate reductase was aligned with FAD domain sequences of other NADH:nitrate reductases, NADH:cytochrome b5 reductases, NADPH:nitrate reductases, ferredoxin:NADP+ reductases, NADPH:cytochrome P-450 reductases, NADPH:sulfite reductase flavoproteins, and Bacillus megaterium cytochrome P-450BM-3. In this multiple alignment, 14 amino acid residues are invariant, which suggests these proteins are members of a family of flavoenzymes. Secondary structure elements of the structural model of spinach ferredoxin:NADP+ reductase were used to predict the secondary structure of squash nitrate reductase and the other related flavoenzymes in this family. We suggest that this family of flavoenzymes, nearly all of which reduce a hemoprotein, be called "flavoprotein pyridine nucleotide cytochrome reductases."     

'Multimodal' kinetics: Cyanobacterial nitrate reductase and other enzyme, transport and binding systems

Concentration‐dependence data for nitrate reductase (EC 1.7.99.4) from heterocystous, nitrogen‐fixing cyanobacteria (J. Martín‐Nieto, E. Flores and A. Herrero, 1992. Plant Physiol. 100: 157–163) have been interpreted most plausibly to reflect the operation of a single enzyme with two independent catalytic sites. However, data from a total of 30 experiments (published as well as unpublished) are, overall, much better ( P < 0.0001) represented according to a 'multimodal' kinetic model rather than as due to two separate sites. This new term is introduced to refer to enzyme systems displaying multiple concentration‐dependent phases separated by sharp inflections. This phenomenon is taken to reflect the operation of a single catalytic site undergoing discontinuous conformational transitions and thus able to function in distinct kinetic 'modes'. Moreover, plots of log K_m versus log V_max in these kinetic systems are perfectly linear, as also previously found for multiphasic plant uptake systems. The same multi‐mode kinetic behavior is exhibited by a wide variety of enzyme, uptake and ligand‐binding systems from plants, animals and microorganisms, including monomeric proteins purified to homogeneity. Multimodal kinetics thus constitute a widespread, albeit largely unrecognized, phenomenon in nature.     

Competition between C-terminal tyrosine and nicotinamide modulates pyridine nucleotide affinity and specificity in plant ferredoxin-NADP(+) reductase

Chloroplast ferredoxin-NADP(+) reductase has a 32,000-fold preference for NADPH over NADH, consistent with its main physiological role of NADP(+) photoreduction for de novo carbohydrate biosynthesis. Although it is distant from the 2'-phosphoryl group of NADP(+), replacement of the C-terminal tyrosine (Tyr(308) in the pea enzyme) by Trp, Phe, Gly, and Ser produced enzyme forms in which the preference for NADPH over NADH was decreased about 2-, 10-, 300-, and 400-fold, respectively. Remarkably, in the case of the Y308S mutant, the k(cat) value for the NADH-dependent activity approached that of the NADPH-dependent activity of the wild-type enzyme. Furthermore, difference spectra of the NAD(+) complexes revealed that the nicotinamide ring of NAD(+) binds at nearly full occupancy in the active site of both the Y308G and Y308S mutants. These results correlate well with the k(cat) values obtained with these mutants in the NADH-ferricyanide reaction. The data presented support the hypothesis that specific recognition of the 2'-phosphate group of NADP(H) is required but not sufficient to ensure a high degree of discrimination against NAD(H) in ferredoxin-NADP(+) reductase. Thus, the C-terminal tyrosine enhances the specificity of the reductase for NADP(H) by destabilizing the interaction of a moiety common to both coenzymes, i.e. the nicotinamide.     

Expression of a novel P275L variant of NADH:cytochrome b5 reductase gives functional insight into the conserved motif important for pyridine nucleotide binding

The clinical disorder of recessive congenital methemoglobinemia (RCM, OMIN 250800) is associated with mutations in NADH:cytochrome b5 reductase (cb5r) and manifests as cyanosis from birth. Screening a cyanotic infant indicated elevated methemoglobin levels and decreased cb5r activity suggesting RCM. Sequencing the DIA1 gene encoding cb5r revealed a novel mutation, C27161T (NCBI accession number: NT_011520), resulting in replacement of proline at amino acid 275 with leucine (P275L). To understand how this mutation would affect cb5r's function, the P275L variant was expressed in a heterologous expression system and spectroscopic, thermodynamic, and thermostability studies were performed. The leucine substitution at residue 275 was found to significantly decrease the affinity towards the physiological reducing substrate, NADH, without affecting the activity of the P275L variant. From the rat model, residue 275 is predicted to be part of a conserved "CGPPPM" motif important for the binding and correct positioning of the NADH reducing substrate. Thus P275 influences the interaction with NADH which was confirmed by the change in affinity towards the physiological reducing substrate.     

Mutagenesis of Glycine 179 modulates both catalytic efficiency and reduced pyridine nucleotide specificity in cytochrome b5 reductase

Cytochrome b5 reductase (cb5r), a member of the ferredoxin:NADP+ reductase family of flavoprotein transhydrogenases, catalyzes the NADH-dependent reduction of cytochrome b5. Within this family, a conserved "GxGxxP" sequence motif has been implicated in binding reduced pyridine nucleotides. However, Glycine 179, a conserved residue in cb5r primary structures, precedes this six-residue "180GxGxxP185" motif that has been identified as binding the adenosine moiety of NADH. To investigate the role of G179 in NADH complex formation and NAD(P)H specificity, a series of rat cb5r variants were generated, corresponding to G179A, G179P, G179T, and G179V, recombinantly expressed in Escherichia coli and purified to homogeneity. Each mutant protein was found to incorporate FAD in a 1:1 cofactor/protein stoichiometry and exhibited absorption and CD spectra that were identical to those of wild-type cb5r, indicating both correct protein folding and similar flavin environments, while oxidation-reduction potentials for the FAD/FADH2 couple (n = 2) were also comparable to the wild-type protein (E(o)' = -272 mV). All four mutants showed decreased NADH:ferricyanide reductase activities, with kcat decreasing in the order WT > G179A > G179P > G179T > G179V, with the G179V variant retaining only 1.5% of the wild-type activity. The affinity for NADH also decreased in the order WT > G179A > G179P > G179T > G179V, with the Km(NADH) for G179V 180-fold greater than that of the wild type. Both Ks(H4NAD) and Ks(NAD+) values confirmed that the G179 mutants had both compromised NADH- and NAD+-binding affinities. Determination of the NADH/NADPH specificity constant for the various mutants indicated that G179 also participated in pyridine nucleotide selectivity, with the G179V variant preferring NADPH approximately 8000 times more than wild-type cb5r. These results demonstrated that, while G179 was not critical for either flavin incorporation or maintenance of the appropriate flavin environment in cb5r, G179 was required for both effective NADH/NADPH selectivity and to maintain the correct orientation and position of the conserved cysteine in the proline-rich "CGpppM" motif that is critical for optimum NADH binding and efficient hydride transfer.     

In vitro reconstitution of NADH: nitrate reductase in nitrate reductase-deficient mutants of barley

Summary In vitro complementation of the nitrate reductase-deficient barley mutant nar2a extracts with molybdenum cofactor from commercial xanthine oxidase resulted in reactivation of NADH: nitrate reductase activity. Maximum reactivation was achieved with 7.5 g/ml xanthine oxidase (final concentration), 10 mM glutathione (final concentration) and incubation for 30 min at room temperature (ca. 25°C). This in vitro complementation assay was used to determine the presence of functional apoprotein and molybdenum cofactor in 12 nitrate reductase-deficient barley mutants. Extracts of all nar1 alleles contained functional molybdenum cofactor (complemented with nar2a) but they lacked functional apoprotein (did not complement with molybdenum cofactor from xanthine oxidase). The nar2a, nar3a and nar3b extracts were able to donate functional apoprotein, but were poor sources of functional molybdenum cofactor. These data are in agreement with our previous assignment of nar1 to the barley NADH: nitrate reductase structural locus and nar2 and nar3 to molybdenum cofactor functions. Wild type cv. Steptoe barley seedlings grown in the absence of nitrate and lacking nitrate reductase activity contained low levels of molybdenum cofactor. Nitrate induction resulted in a several-fold increase in the measurable molybdenum cofactor levels that was correlated with the increase in nitrate reductase activity.Scientific Paper No. 6839. College of Agriculture Research Center, Washington State University, Pullman. Project Nos. 0430 and 0233. This work was supported in part by National Science Foundation Grant PCM 81-19096 and USDA Competitive Research Grant 82-CRCR-1-1112     

Unusual regulatory nitrate reductase activity in cotyledons of Brassica napus seedlings: enhancement of nitrate reductase activity by ammonium supply

The effect of supplying either nitrate or ammonium on nitrate reductase activity (NRA) was investigated in Brassica napus seedlings. In roots, nitrate reductase activity (NRA) increased as a function of nitrate content in tissues and decreased when ammonium was the sole nitrogen source. Conversely, in the shoots (comprising the cotyledons and hypocotyl), NRA was shown to be independent of nitrate content. Moreover, when ammonium was supplied as the sole nitrogen source, NRA in the shoots was surprisingly higher than under nitrate supply and increased as a function of the tissue ammonium content. Under 15 mM of exogenous ammonium, the NRA was up to 2.5-fold higher than under nitrate supply after 6 d of culture. The NR mRNA accumulation under ammonium nutrition was 2-fold higher than under nitrate supply. The activation state of NR in shoots was especially high compared with roots: from nearly 80% under nitrate supply it reached 94% under ammonium. This high NR activation state under ammonium supply could be the consequence of the slight acidification observed in the shoot tissue. The effect of ammonium on NRA was only observed in cotyledons and when more than 3 mM ammonium was supplied. No such NRA increase was evident in the roots or in foliar discs. Addition of 1 mM nitrate under ammonium nutrition halved NRA and decreased the ammonium content in shoots. Thus, this unusual NRA was restricted to seedling cotyledons when nitrate was lacking in the nitrogen source.     

Involvement of lysine residue in the nucleotide binding of pigeon liver malic enzyme: modification with affinity label periodate-oxidized nadp

• 1.1. Periodate-oxidized NADP, a competitive inhibitor of malic enzyme with respect to NADP. inactivate the enzyme in mild conditions.
• 2.2. The inactivation is due to the modification of an essential lysine residue.
• 3.3. Two molecules of reagent were found to be incorporated into the enzyme tetramer after extensive modification.
• 4.4. Complete protection of malic enzyme from the oxidized NADP inactivation was afforded by NADP and its analogues.
• 5.5. The modified enzyme showed increased apparent Michaelis constant for the nucleotide coenzymes but the maximum velocity was decreased.
• 6.6. The binding between the modified enzyme and NADPH was impaired.