Direct transfer of molybdopterin cofactor to aponitrate reductase from a carrier protein in Chlamydomonas reinhardtii.

A Chlamydomonas reinhardtii molybdenum cofactor (MoCo)-carrier protein (CP), capable of reconstituting nitrate reductase activity with apoprotein from the Neurospora crassa mutant nit-1, was subjected to experiments of diffusion through a dialysis membrane and gel filtration. CP bonded firmly MoCo and did not release it efficiently unless aponitrate reductase was present in the incubation mixture. Stability of MoCo bound to CP against air and heat was very similar to that of free-MoCo released from milk xanthine oxidase. Our data strongly suggest that MoCo is directly transferred from CP to aponitrate reductase to form an active enzyme.     

Quantitation of molybdopterin oxidation product in wild-type and molybdenum cofactor deficient mutants of Chlamydomonas reinhardtii.

A simple and reliable procedure of oxidation of molybdenum cofactor (MoCo) from molybdoenzymes by autoclaving samples at 120 degrees C for 20 min yielded a single predominant fluorescent species that could be quantitatively determined by reverse phase high performance liquid chromatography. This method allowed detection and quantitation of molybdopterin in cell-free extracts of the green alga Chlamydomonas reinhardtii. The MoCo oxidation product from C. reinhardtii has the same chromatographic and spectral properties as that of milk xanthine oxidase and chicken liver sulfite oxidase. The oxidized species was also detected in molybdenum cofactor mutants of Chlamydomonas reinhardtii defective at the nit-3, nit-4, nit-5/nit-6 and nit-7 loci, which strongly suggests that active molybdenum cofactor itself is not directly involved in the control of its own biosynthetic pathway.     

Two different carriers transport both ammonium and methylammonium in Chlamydomonas reinhardtii

A new methylammonium-resistant mutant strain from Chlamydomonas reinhardtii, henceforth termed 2172 (ma-2), has been isolated. This mutant is affected in a single mendelian gene different from and linked to the ma-1 locus which is defective in the methylammonium-resistant mutant 2170. Both mutations in ma-1 (2170) and ma-2 (2172) are linked to the nit-1 gene coding for the nitrate reductase apoenzyme. Mutant 2172 is affected in methylammonium but not in ammonium uptake capacity and shows derepressed nitrate and nitrite reductase activities in media containing nitrate plus methylammonium but not in nitrate plus ammonium media. The following two enzymatic components for the transport of both ammonium and methylammonium in wild-type cells have been identified: component 1, with high Vmax and K values, which is constitutive, and component 2, with low Vmax and K values, which is ammonium-repressible. Mutant 2170 lacks component 1 whereas mutant 2172 lacks component 2 for both methylammonium and ammonium transport. From genetic and kinetic evidences we conclude that in C. reinhardtii two different carriers are responsible for the transport of both ammonium and methylammonium and that methylammonium (ammonium) transport is a reversible process probably inhibited by the intracellular ammonium which, in turn, regulates nitrate and nitrite reductase levels.     

Regulation by ammonium of nitrate and nitrite assimilation in Chlamydomonas reinhardtii

The inhibitor of mRNA synthesis, 6-methylpurine, inhibited nitrate reductase derepression in either ammonium-grown or methylammonium-treated wild-type cells of Chlamydomonas reinhardtii, but not in nitrogen-starved cells. In contrast, 6-methylpurine did not inhibit nitrate reductase synthesis in the methylammonium-resistant mutant 2170 (ma-1) either grown on ammonium, treated with methylammonium or nitrogen starved, but did inhibit the continuous synthesis of nitrate reductase, which required the presence of nitrate in the media. In both wild-type and mutant 2170 grown on ammonium and transferred to nitrate media, cycloheximide immediately prevented nitrate reductase derepression when added either at the beginning or at different times of induction treatment. Unlike wild-type cells, mutant 2170 was able to take up either nitrate or nitrite simultaneously with ammonium in whose presence nitrate and nitrite reductases were synthesized. However, synthesis of nitrate reductase was progressively inhibited in the mutant cells when the intracellular ammonium levels were raised as a result of an increase in either the external pH or the extracellular ammonium concentrations. The results rule out the existence of maturase-like proteins in Chlamydomonas and indicate that ammonium has a double effect on the regulation of nitrate reductase synthesis: (a) it prevents nitrate reductase mRNA production; and (b) it controls negatively the expression of this mRNA.     

Induction and Repression of Nitrate Reductase in Neurospora crassa

Synthesis of wild-type Neurospora crassa assimilatory nitrate reductase is induced in the presence of nitrate ions and repressed in the presence of ammonium ions. Effects of several Neurospora mutations on the regulation of this enzyme are shown: (i) the mutants, nit-1 and nit-3, involving separate lesions, lack reduced nicotinamide adenine dinucleotide (NADPH)-nitrate reductase activity and at least one of three other activities associated with the wild-type enzyme. The two mutants do not require the presence of nitrate for induction of their aberrant nitrate reductases and are constitutive for their component nitrate reductase activities in the absence of ammonium ions. (ii) An analog of the wild-type enzyme (similar to the nit-1 enzyme) is formed when wild type is grown in a medium in which molybdenum has been replaced by vanadium or tungsten; the resulting enzyme lacks NADPH-nitrate reductase activity. Unlike nit-1, wild type produced this analog only in the presence of nitrate. Contaminating nitrate does not appear to be responsible for the observed mutants' activities. Nitrate reductase is proposed to be autoregulated. (iii) Mutants (am) lacking NADPH-dependent glutamate dehydrogenase activity partially escape ammonium repression of nitrate reductase. The presence of nitrate is required for the enzyme's induction. (iv) A double mutant, nit-1 am-2, proved to be an ideal test system to study the repressive effects of nitrogen-containing metabolites on the induction of nitrate reductase activity. The double mutant does not require nitrate for induction of nitrate reductase, and synthesis of the enzyme is not repressed by the presence of high concentrations of ammonium ions. It is, however, repressed by the presence of any one of six amino acids. Nitrogen metabolites (other than ammonium) appear to be responsible for the mediation of "ammonium repression."     

Regulation of Nitrate Reductase in Neurospora crassa: Stability In Vivo

Nicotinamide adenine dinucleotide phosphate, reduced form (NADPH)-nitrate reductase and its related enzyme activities, NADPH-cytochrome c reductase and reduced benzyl viologen-nitrate reductase, are all induced following the transfer of ammonia-grown wild-type Neurospora mycelia to nitrate medium. After nitrate reductase is induced to the maximal level, the addition of an ammonium salt to, or the removal of nitrate from, the cultures results in a rapid inactivation of nitrate reductase and its two partial component activities. This rapid inactivation is slowed down by the protein synthesis inhibitor, cycloheximide. Experiments on the mixing of extracts in vitro rule out the presence of an inhibitor of nitrate reductase in free form in extracts containing inactivated nitrate reductase. Ammonia does not inhibit the uptake of nitrate by the mycelia. Inactivation of nitrate reductase in vivo by ammonia depends on the concentration of the ammonium salt and is not reversed by increasing the nitrate concentration of the medium. The nitrate-inducible NADPH-cytochrome c reductase activity and reduced benzyl viologen-nitrate reductase activity respectively of the nitrate-nonutilizing mutants nit-1 and nit-3 are not inactivated in vivo by the addition of an ammonium salt or the withdrawal of nitrate. This finding suggests that the integrity of the nitrate reductase complex is required for the in vivo inactivation of nitrate reductase and its associated activities.     

Nitrate Reductase Regulates Expression of Nitrite Uptake and Nitrite Reductase Activities in Chlamydomonas reinhardtii

In Chlamydomonas reinhardtii mutants defective at the structural locus for nitrate reductase (nit-1) or at loci for biosynthesis of the molybdopterin cofactor (nit-3, nit-4, or nit-5 and nit-6), both nitrite uptake and nitrite reductase activities were repressed in ammonium-grown cells and expressed at high amounts in nitrogen-free media or in media containing nitrate or nitrite. In contrast, wild-type cells required nitrate induction for expression of high levels of both activities. In mutants defective at the regulatory locus for nitrate reductase (nit-2), very low levels of nitrite uptake and nitrite reductase activities were expressed even in the presence of nitrate or nitrite. Both restoration of nitrate reductase activity in mutants defective at nit-1, nit-3, and nit-4 by isolating diploid strains among them and transformation of a structural mutant upon integration of the wild-type nit-1 gene gave rise to the wild-type expression pattern for nitrite uptake and nitrite reductase activities. Conversely, inactivation of nitrate reductase by tungstate treatment in nitrate, nitrite, or nitrogen-free media made wild-type cells respond like nitrate reductase-deficient mutants with respect to the expression of nitrite uptake and nitrite reductase activities. Our results indicate that nit-2 is a regulatory locus for both the nitrite uptake system and nitrite reductase, and that the nitrate reductase enzyme plays an important role in the regulation of the expression of both enzyme activities.     

Molybdenum cofactor amounts in Chlamydomonas reinhardtii depend on the Nit5 gene function related to molybdate transport

Strain 21gr from Chlamydomonas reinhardtii is a cryptic mutant defective in the Nit5 gene related to the biosynthesis of molybdenum cofactor (MoCo). In spite of this mutation, this strain has active MoCo and can grow on nitrate media. In genetic crosses, the Nit5 mutation cosegregated with a phenotype of resistance to high concentrations of molybdate and tungstate. Molybdate/tungstate toxicity was much higher in nitrate than in ammonium media. Strain 21gr showed lower amounts of MoCo activity than the wild type both when grown in nitrate and after growth in ammonium and nitrate induction. However, nitrate reductase (NR) specific activity was similar in wild type and 21gr cells. Tungstate, either at nanomolar concentrations in nitrate media or at micromolar concentrations during growth in ammonium and nitrate induction, strongly decreased MoCo and NR amounts in wild‐type cells but had a slight effect in 21gr cells. Molybdate uptake activity of ammonium‐grown cells from both the wild‐type and 21gr strains was small and blocked by sulphate 0·3 mM . However, cells from nitrate medium showed a molybdate uptake activity insensitive to sulphate. This uptake activity was much higher and more sensitive to inhibition by tungstate in the wild type than in strain 21gr. These results suggest that strain 21gr has a high affinity and low capacity molybdate transport system able to discriminate efficiently tungstate, and lacks a high capacity molybdate/tungstate transport system, which operates in wild‐type cells upon nitrate induction. This high capacity molybdate transport system would account for both the stimulating effect of molybdate on MoCo amounts and the toxic effects of tungstate and molybdate when present at high concentrations.     

Chlamydomonas reinhardtii strains expressing nitrate reductase under control of the cabII-1 promoter: isolation of chlorate resistant mutants and identification of new loci for nitrate assimilation

The Chlamydomonas reinhardtii strain Tx11-8 is a transgenic alga that bears the nitrate reductase gene (Nia1) under control of the CabII-1 gene promoter (CabII-1-Nia1). Approximately nine copies of the chimeric CabII-1-Nia1 gene were found to be integrated in this strain and to confer a phenotype of chlorate sensitivity in the presence of ammonium. We have used this strain for the isolation of spontaneous chlorate resistant mutants in the presence of ammonium that were found to be defective at loci involved in MoCo metabolism and light-dependent growth in nitrate media. Of a total of 45 mutant strains analyzed first, 44 were affected in the MoCo activity (16 Nit⁻, unable to grow in nitrate, and 28 Nit⁺, able to grow in nitrate). All the Nit⁻ strains lacked MoCo activity. Diploid complementation of Nit⁻, MoCo⁻ strains with C. reinhardtii MoCo mutants and genetic analysis indicated that some strains were defective at known loci for MoCo biosynthesis, while three strains were defective at two new loci, hereafter named Nit10 and Nit11. The other 28 Nit⁺ strains showed almost undetectable MoCo activity or activity was below 20% of the parental strain. Second, only one strain (named 23c⁺) showed MoCo and NR activities comparable to those in the parental strain. Strain 23c⁺ seems to be affected in a locus, Nit12, required for growth in nitrate under continuous light. It is proposed that this locus is required for nitrate/chlorate transport activity. In this work, mechanisms of chlorate toxicity are reviewed in the light of our results.     

Evidence for MoeA-dependent formation of the molybdenum cofactor from molybdate and molybdopterin in<Emphasis Type="Italic"> Escherichia coli</Emphasis>

The function of the MoeA protein in the biosynthesis of the molybdenum cofactor (MoCo) was analyzed in vitro, using purified His(6)-MoeA from Escherichia coli, molybdopterin (MPT) isolated from buttermilk xanthine oxidase and molybdate. The formation of MoCo was monitored by the reconstitution of nitrate reductase activity in extracts of the Neurospora crassa nit-1 mutant. Formation of MoCo from MPT and molybdate required MoeA and L-cysteine or glutathione. The reaction proceeded at micromolar molybdate levels and was time- and MoeA concentration-dependent. A physical interaction between MoeA and MPT was demonstrated by HPLC analysis of MoeA-bound MPT.     

Identification of the molybdenum cofactor in chlorate-resistant mutants of Escherichia coli.

Experiments were performed to determine whether defects in molybdenum cofactor metabolism were responsible for the pleiotropic loss of the molybdoenzymes nitrate reductase and formate dehydrogenase in chl mutants of Escherichia coli. In wild-type E. coli, molybdenum cofactor activity was present in both the soluble and membrane-associated fractions when the cells were grown either aerobically or anaerobically, with and without nitrate. Molybdenum cofactor in the soluble fraction decreased when the membrane-bound nitrate reductase and formate dehydrogenase were induced. In the chl mutants, molybdenum cofactor activity was found in the soluble fraction of chlA, chlB, chlC, chlD, chlE, and chlG, but only chlB, chlC, chlD, and chlG expressed cofactor activity in the membrane fraction. The defect in the chlA mutants which prevented incorporation of the soluble cofactor into the membrane also caused the soluble cofactor to be defective in its ability to bind molybdenum. This cofactor was not active in the absence of molybdate, and it required at least threefold more molybdate than did the wild type in the Neurospora crassa nit-1 complementation assay. However, the cofactor from the chlA strain mediated the dimerization of the nit-1 subunits in the presence and absence of molybdate to yield the 7.9S dimer. Growth of chlA mutants in medium with increased molybdate did not repair the defect in the chlA cofactor nor restore the molybdoenzyme activities. Thus, molybdenum cofactor was synthesized in all the chl mutants, but additional processing steps may be missing in chlA and chlE mutants for proper insertion of cofactor in the membrane.     

Regulation of nitrate reductase of Neurospora at the level of transcription and translation

The presence of nitrate is required for the induced synthesis of NADPH-nitrate reductase and its related partial activity Benzyl Viologen-nitrate reductase in a wild-type strain of Neurospora. In nit-3, a mutant lacking complete NADPH-nitrate reductase activity but retaining the partial activity Benzyl Viologen-nitrate reductase, the presence of nitrate ions is not required for the de-repressed synthesis of the latter enzyme. The accumulation of the capacity to synthesize nitrate reductase, and the related Benzyl Viologen-nitrate reductase, in the absence of protein synthesis does not require nitrate in the normal strain or in strain nit-3. Ammonia antagonizes the accumulation of this capacity in both strains. Nitrate is required for the synthesis of nitrate reductase and related activities from presumedly preformed mRNA in the wild-type strain. Nitrate is not required for the comparable function in strain nit-3. Ammonia appears to stop the synthesis of nitrate reductase and related activities from presumedly preformed mRNA in the wild-type strain and in strain nit-3. The effects of nitrate, or ammonia and of no nitrogen source on the induced synthesis of nitrate reductase cannot be explained on the basis of the effects of the different nitrogen sources on general synthesis of RNA or of protein.     

A study of the nitrate and nitrite discharge from the mutant cells of Neurospora crassa lacking nitrate and nitrite reductase activities

The Neurospora crassa mutants nit-2 (lacking both nitrite and nitrate reductases) and nit-6 (lacking nitrite reductase) grown in the medium with ammonium chloride as a sole source of nitrogen discharged nitrate and nitrite ions into culture medium. For nit-2, the content of nitrate exceeded that of nitrite in both the homogenate of fungal cells and growth medium; moreover, this difference was more pronounced in the culture medium. Unlike nit-2, the content of nitrite in the cultivation medium of the nit-6 mutant irradiated with visible light for 30 min during the lag phase of carotenogenesis photoinduction displayed a trend of increase as compared with the dark control. Further (to 240 min) irradiation of cells, i.e., irradiation during biosynthesis of carotenoid pigments, leveled this difference.     

Characterization of molybdenum cofactor from Escherichia coli.

Molybdenum cofactor activity was found in the soluble fraction of cell-free extracts of Escherichia coli grown aerobically in media supplemented with molybdate. Cofactor was detected by its ability to complement the nitrate reductase-deficient mutant of Neurospora crossa, nit-1, resulting in the vitro formation of nitrate reductase activity. Acid treatment of E. coli extracts was not required for release of cofactor activity. Cofactor was able to diffuse through a membrane of nominal 2,000-molecular-weight cutoff and was insensitive to trypsin. The cofactor was associated with a carrier molecule (approximately 40,000 daltons) during gel filtration and sucrose gradient centrifugation, but was easily removed from the carrier by dialysis. The carrier molecule protected the cofactor from inactivation by heat or oxygen. E. coli grown in molybdenum-free media, without and with tungsten, synthesized a metal-free "empty" cofactor and its tungsten analog, respectively, both of which were subsequently activated by the addition of molybdate. Empty and tungsten-containing cofactor complemented the nitrate reductase subunits in the nit-1 extract, forming inactive, but intact, 7.9S nitrate reductase. Addition of molybdate to the enzyme complemented in this manner restored nitrate reductase activity.     

Regulation of aldehyde oxidase and nitrate reductase in roots of barley (Hordeum vulgare L.) by nitrogen source and salinity

The molybdenum cofactor (MoCo) is a component of aldehyde oxidase (AO EC 1.2.3.1), xanthine dehydrogenase (XDH EC 1.2.1.37) and nitrate reductase (NR, EC 1.6.6.1). The activity of AO, which catalyses the last step of the synthesis of abscisic acid (ABA), was studied in leaves and roots of barley (Hordeum vulgare L.) plants grown on nitrate or ammonia with or without salinity. The activity of AO in roots was enhanced in plants grown with ammonium while nitrate-grown plants exhibited only traces. Root AO in barley was enhanced by salinity in the presence of nitrate or ammonia in the nutrient medium while leaf AO was not significantly affected by the nitrogen source or salinity of the medium.Salinity and ammonium decreased NR activity in roots while increasing the overall MoCo content of the tissue. The highest level of AO in barley roots was observed in plants grown with ammonium and NaCl, treatments that had only a marginal effect on leaf AO. ABA concentration in leaves of plants increased with salinity and ammonium.Keywords: ABA, aldehyde oxidase, ammonium, nitrate, salinity.      

Identification and Characterization of a Nitrate Transporter Gene in Neurospora crassa

The Neurospora crassa genome database was searched for sequence similarity to crnA, a nitrate transporter in Aspergillus nidulans. A 3.9-kb fragment (contig 3.416, subsequence 183190-187090) was cloned by PCR. The gene coding for this nitrate transporter was termed nit-10. The nit-10 gene specifies a predicted polypeptide containing 541 amino acids with a molecular mass of 57 kDa. In contrast to crnA, which is clustered together with niaD, encoding nitrate reductase, and niiA, encoding nitrite reductase, nit-10 is not linked to nit-3 (nitrate reductase), nit-6 (nitrite reductase), or to nit-2, nit-4 (both are positive regulators of nit-3), or nmr (negative regulator of nit-3) in Neurospora crassa. A nit-10 rip mutant failed to grow in the medium when nitrate (< 10 mM) was used as the sole nitrogen source, but grew similarly to wild type when nitrate concentration was 10 mM or higher. In addition, it showed strong sensitivity to cesium in the presence of nitrate and resistance to chlorate in the presence of alanine, proline, or hypoxanthine. The expression of nit-10 required nitrate induction and was subject to repression by nitrogen metabolites such as glutamine. Expression of nit-10 also required functional products of nit-2 and nit-4. The half-life of nit-10 mRNA was determined to be approximately 2.5 min.     

Functional analysis of molybdopterin biosynthesis in mycobacteria identifies a fused molybdopterin synthase in Mycobacterium tuberculosis

Most mycobacterial species possess a full complement of genes for the biosynthesis of molybdenum cofactor (MoCo). However, a distinguishing feature of members of the Mycobacterium tuberculosis complex is their possession of multiple homologs associated with the first two steps of the MoCo biosynthetic pathway. A mutant of M. tuberculosis lacking the moaA1-moaD1 gene cluster and a derivative in which moaD2 was also deleted were significantly impaired for growth in media containing nitrate as a sole nitrogen source, indicating a reduced availability of MoCo to support the assimilatory function of the MoCo-dependent nitrate reductase, NarGHI. However, the double mutant displayed residual respiratory nitrate reductase activity, suggesting that it retains the capacity to produce MoCo. The M. tuberculosis moaD and moaE homologs were further analyzed by expressing these genes in mutant strains of M. smegmatis that lacked one or both of the sole molybdopterin (MPT) synthase-encoding genes, moaD2 and moaE2, and were unable to grow on nitrate, presumably as a result of the loss of MoCo-dependent nitrate assimilatory activity. Expression of M. tuberculosis moaD2 in the M. smegmatis moaD2 mutant and of M. tuberculosis moaE1 or moaE2 in the M. smegmatis moaE2 mutant restored nitrate assimilation, confirming the functionality of these genes in MPT synthesis. Expression of M. tuberculosis moaX also restored MoCo biosynthesis in M. smegmatis mutants lacking moaD2, moaE2, or both, thus identifying MoaX as a fused MPT synthase. By implicating multiple synthase-encoding homologs in MoCo biosynthesis, these results suggest that important cellular functions may be served by their expansion in M. tuberculosis.     

nit 7: A New Locus for Molybdopterin Cofactor Biosynthesis in the Green Alga Chlamydomonas reinhardtii

Two new nitrate reductase-deficient mutants from Chlamydomonas reinhardtii have been genetically and biochemically characterized. Both H1 and F23 mutants carry single recessive allelic mutations that map at a new locus designated nit-7. This locus is unlinked to the other six nit loci related to the nitrate assimilation pathway in C. reinhardtii. Both mutant alleles H1 and F23 lack an active molybdopterin cofactor, the activity of which is restored neither in vitro nor in vivo by high concentrations of molybdate. Nitrate reductase subunits in these mutants seem to assemble, although not in a stable form, in a high molecular weight complex and, as in other molybdenum cofactor-defective mutants of C. reinhardtii, they cannot reconstitute nitrate reductase activity with an active molybdenum cofactor source from extracts of ammonium-grown cells. The results suggest that nit-7 mutants are defective in molybdopterin biosynthesis. They do produce some precursor(s) that are capable of binding to nitrate reductase subunits.