Stimulation of nitrate reductase activity of the salt-tolerant yeast Rhodotorula glutinis by tungsten in the presence of molybdenum

Tungsten in the presence of molybdenum stimulates nitrate reductase activity and growth of the salt-tolerant yeast Rhodotorula glutinis on medium with nitrates. Tungsten is not incorporated in proteins possessing nitrate reductase activity. A significant increase in molybdenum cofactor in cells grown on medium with equimolar amounts of molybdenum and tungsten may relate to the stimulatory action of tungsten.     

Effects of molybdenum and tungsten on induction of nitrate reductase and formate dehydrogenase in wild type and mutant Paracoccus denitrificans

Molybdenum is required for induction of nitrate reductase and of NAD-linked formate dehydrogenase activities in suspensions of wild type Paracoccus denitrificans; tungsten prevents the development of these enzyme activities. The wild type forms a membrane protein M _r150,000 when incubated with tungsten and inducers of nitrate reductase and this is presumed to represent an inactive form of the enzyme. Suspensions of mutant M-1 did not develop nitrate reductase or formate dehydrogenase activities but the membrane protein M _r150,000 was formed under all conditions tested, including without inducers and without molybdenum. Analysis of membranes, solubilized with deoxycholate, by polyacrylamide gel electrophoresis under nondenaturing conditions showed that the mutant protein had similar electrophoretic mobility to the active nitrate reductase formed by the wilde type. Autoradiography of preparations from cells incubated with ⁵⁵Fe showed that the mutant and wild type proteins contained iron. However, in similar experiments with ⁹⁹Mo, incorporation of molybdenum into the mutant protein was not detectable.We conclude that mutant M-1 is defective in one or more steps required to process molybdenum for incorporation into molybdoenzymes. This failure affects the normal regulation of nitrate reductase protein with respect to the role of inducers.Non-Standard Abbreviations DOC deoxycholate - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Differential effect of tungsten on the development of endogenous and nitrate-induced nitrate reductase activities in soybean leaves

The effect of tungsten on the development of endogenous and nitrate-induced NADH- and FMNH₂-linked nitrate reductase activities in primary leaves of 10-day-old soybean (Glycine max [L.] Merr.) seedlings was studied. The seedlings were grown with or without exogenous nitrate. High levels of endogenous nitrate reductase activities developed in leaves of seedlings grown without nitrate. However, no endogenous nitrite reductase activity was detected in such seedlings. The FMNH₂-linked nitrate reductase activity was about 40% of NADH-linked activity. Tungsten had little or no effect on the development of endogenous NADH- and FMNH₂-linked nitrate reductase activities, respectively. By contrast, in nitrate-grown seedlings, tungsten only inhibited the nitrate-induced portion of NADH-linked nitrate reductase activity, whereas the FMNH₂-linked activity was inhibited completely. Tungsten had no effect on the development of nitrate-induced nitrite reductase activity. The complete inhibition of FMNH₂-linked nitrate reductase activity by tungsten in nitrate-grown plants was apparently an artifact caused by the reduction of nitrite by nitrite reductase in the assay system. The results suggest that in soybean leaves either the endogenous nitrate reductase does not require molybdenum or the molybdenum present in the seed is preferentially utilized by the enzyme complex as compared to nitrate-induced nitrate reductase.     

Molybdenum and tungsten in Campylobacter jejuni: their physiological role and identification of separate transporters regulated by a single ModE-like protein

Campylobacter jejuni is an important human pathogen that causes millions of cases of food-borne enteritis each year. The C. jejuni respiratory chain is highly branched and contains at least four enzymes predicted to contain a m etal binding pt erin (MPT), with the metal being either molybdenum or tungsten. Also predicted are two separate transport systems, one for molybdenum encoded by modABC and a second for tungsten encoded by tupABC . Both transport systems were mutated and the activities of the four predicted MPT-containing enzymes were assayed in the presence of molybdenum and tungsten in wild-type and mod and tup backgrounds. Results indicate that mod is primarily a molybdenum transporter that can also transport tungsten, while tup is a tungsten-specific transporter. The MPT-containing enzymes nitrate reductase, sulphite oxidase, and SN oxide reductase are strict molybdoenzymes while formate dehydrogenase prefers tungsten. A ModE-like protein regulates both transporters, repressing mod in the presence of both molybdenum and tungsten and tup only in the presence of tungsten. Like other ModE proteins, the C. jejuni ModE binds DNA through a helix–turn–helix DNA binding domain, but unlike other members of the ModE family it does not have a metal binding domain.     

Transport of molybdate in the cyanobacterium Anabaena variabilis ATCC 29413

Heterocyst-forming filamentous cyanobacteria, such as Anabaena variabilis ATCC 29413, require molybdenum as a component of two essential cofactors for the enzymes nitrate reductase and nitrogenase. A. variabilis efficiently transported (99)Mo (molybdate) at concentrations less than 10(-9) M. Competition experiments with other oxyanions suggested that the molybdate-transport system of A. variabilis also transported tungstate but not vanadate or sulfate. Although tungstate was probably transported, tungsten did not function in place of molybdenum in the Mo-nitrogenase. Transport of (99)Mo required prior starvation of the cells for molybdate, suggesting that the Mo-transport system was repressed by molybdate. Starvation, which required several generations of growth for depletion of molybdate, was enhanced by growth under conditions that required synthesis of nitrate reductase or nitrogenase. These data provide evidence for a molybdate storage system in A. variabilis. NtcA, a regulatory protein that is essential for synthesis of nitrate reductase and nitrogenase, was not required for transport of molybdate. The closely related strain Anabaena sp. PCC 7120 transported (99)Mo in a very similar way to A. variabilis.     

Effect of cyanophage N-1 infection on the synthesis and stability ofNostoc muscorum nitrate reductase

The control operative on the nitrate reductase enzyme system of host cyanobacteriumNostoc muscorum was studied after being infected with the cyanophage N-1. Phage infection lifted the host nitrate reductase activity level via accelerating the enzyme synthesis. It was found that the phage-mediated increase in the molybdenum cofactor synthesis was a major contributing factor for apparent elevated nitrate reductase level of the host. This process was inhibited in the presence of erythromycin and tungsten, the inhibitors of protein synthesis and new nitrate reductase synthesis respectively. While the preformed nitrate reductase of healthy cyanobacterium was inhibited by hydrogen peroxide, an oxidizing photosynthetic product, the same enzyme of infected cells remained virtually insensitive to this inhibitor. These data suggest involvement of new nitrate reductase synthesis and its resistance to oxidative inactivation as joint factors controlling the characteristic high enzyme level of host cyanobacterium.     

Regulation of nitrate reductase inRhodobacter capsulatus E1F1

The photosynthetic purple non-sulfur nitrate-assimilating bacteriumRhodobacter capsulatus E1F1 has an adaptive nitrate reductase activity inducible by either nitrate or nitrite and molybdenum traces. Nitrate reductase induction by nitrate did not occur in media with nitrate and ammonium, which showed no effect if nitrite was the inductor instead of nitrate or in the presence ofl-methionine-dl-sulfoximine (MSX) plus nitrate. In vivo, tungstate inhibited nitrate reductase activity, and this was not recovered upon addition of molybdenum unless de novo protein synthesis took place. Nitrate reductase was also repressed in nitrogen-starved cells or after the addition of azaserine to cells growing phototrophically with nitrate. Moreover, higher rates of nitrate reductase induction and nitrite excretion were found in illuminated cells grown with nitrate under air than in those grown under argon.     

In vitro reconstitution of nitrate reductase activity of the Neurospora crassa mutant nit-1: specific incorporation of molybdopterin

The reduced, metal-free pterin of the molybdenum cofactor has been termed molybdopterin. Oxidation of any molybdopterin-containing protein in the presence or absence of iodine yields oxidized molybdopterin derivatives termed Form A and Form B, respectively. Application of these procedures to whole cells and cell extracts has demonstrated the presence of molybdopterin in wild-type Neurospora crassa, and its absence in the cofactor-deficient mutant nit-1. In order to demonstrate that the reconstitution of nitrate reductase activity in nit-1 extracts results from the incorporation of molybdopterin into the apoprotein, active molybdopterin, free of contaminating amino acids or peptides, was isolated from chicken liver sulfite oxidase and used in the reconstitution system. The results show that, during reconstitution, exogenous molybdopterin is specifically incorporated into the nitrate reductase protein, confirming the role of molybdopterin as the organic moiety of the molybdenum cofactor.     

Bacterial expression of the molybdenum domain of assimilatory nitrate reductase: production of both the functional molybdenum-containing domain and the nonfunctional tungsten analog

Assimilatory NADH:nitrate reductase (EC 1.6.6.1), a complex molybdenum-, cytochrome b(557)- and FAD-containing protein, catalyzes the regulated and rate-limiting step in the utilization of inorganic nitrogen by higher plants. To facilitate structure/function studies of the individual molybdenum center, we have developed bacterial expression systems for the heterologous production of the 541 residue amino-terminal, molybdenum center-containing domain of spinach nitrate reductase either as a six-histidine-tagged variant or as a glutathione-S-transferase-tagged fusion protein. Expression of the his-tagged molybdenum domain in Escherichia coli BL21(DE3) cells under anaerobic conditions yielded a 55-kDa domain with a specific activity of 1.5 micromol NO(3)(-) consumed/min/nmol enzyme and with a K(mapp)(NO(3)(-)) of 8 mciroM. In contrast, expression of the molybdenum domain as a GST-tagged fusion protein in E. coli TP1000(MobA(-) strain) cells under aerobic conditions yielded an 85-kDa fusion protein with a specific activity of 10.8 micromol NO(3)(-) consumed/min/nmol enzyme and with a K(mapp)(NO(3)(-)) of 12 microM. Fluorescence analysis indicated that both forms of the molybdenum domain contained the cofactor, MPT, although the MPT content was higher in the GST-fusion domain. Inductively coupled plasma mass spectrometric analysis of both the his-tagged and GST-fusion protein domain samples indicated Mo/protein ratios of 0.44 and 0.93, respectively, confirming a very high level of Mo incorporation in the GST-fusion protein. Expression of the GST-fusion protein in TP1000 cells in the presence of elevated tungsten concentrations resulted in an 85-kDa fusion protein that contained MPT but which was devoid of nitrate-reducing activity. Partial reduction of the molybdenum domain resulted in the generation of an axial Mo(V) EPR species with g values of 1.9952, 1.9693, and 1.9665, respectively, and exhibiting superhyperfine coupling to a single exchangeable proton, analogous to that previously observed for the native enzyme. In contrast, the tungsten-substituted MPT-containing domain yielded a W(V) EPR species with g values of 1.9560, 1.9474, and 1.9271, respectively, with unresolved superhyperfine interaction. NADH:nitrate reductase activity could be reconstituted using the GST-molybdenum domain fusion protein in the presence of the recombinant forms of the spinach nitrate reductase' flavin- and heme-containing domains.     

Mode of action of natural inactivator proteins from corn and rice on a purified assimilatory nitrate reductase

The molecular basis for the action of two natural inactivator proteins, isolated from rice and corn, on a purified assimilatory nitrate reductase has been examined by several physical techniques. Incubation of purified Chlorella nitrate reductase with either rice inactivator protein or corn inactivator protein results in a loss of NADH:nitrate reductase and the associated partial activity, NADH:cytochrome c reductase, but no loss in nitrate-reducing activity with reduced methyl viologen as the electron donor. The molecular weight of the reduced methyl viologen:nitrate reductase species, determined by sedimentation equilibrium in the Beckman airfuge after complete inactivation with rice inactivator protein or with corn inactivator protein, was 595,000 and 283,000, respectively, compared to a molecular weight of 376,000 for the untreated control determined under the same conditions. Two protein peaks were observed after molecular-sieve chromatography on Sephacryl S-300 of nitrate reductase inactivated by corn inactivator protein. The Stokes radii of these fragments were 68 and 24 Å, compared to a value of 81 Å for untreated nitrate reductase. The large fragment contained molybdenum and heme but no flavin, and had nitrate-reducing activity with reduced methyl viologen as electron donor. The small fragment contained FAD but had no NADH:cytochrome c reductase or nitrate-reducing activities. Molecular weights determined by sodium dodecyl sulfate-gel electrophoresis were 67,000 and 28,000 for the large and small fragments, respectively, compared to a subunit molecular weight of 99,000 determined for the untreated control. No change in subunit molecular weight of nitrate reductase after inactivation by rice inactivator protein was observed. These results indicate that rice inactivator protein acts by binding to nitrate reductase. The stoichiometry of binding is 1–2 molecules of rice inactivator protein to one tetrameric molecule of nitrate reductase. Corn inactivator protein, in contrast, acts by cleavage of a M_r 30,000 fragment from nitrate reductase which is associated with FAD. The remaining fragment is a tetramer of M_r 70,000 subunits which retains nitrate-reducing activity and contains molybdenum and heme but has no NADH:dehydrogenase activity. The action of rice inactivator protein was partially prevented by NADH and completely prevented by a combination of NADH and cyanide, while the action of corn inactivator protein was not significantly affected by these effectors.     

In Vitro Incorporation of Molybdate into Demolybdoproteins in Escherichia coli

When Escherichia coli was grown in the presence of tungstate, inactive forms of two molybdoenzymes, nitrate reductase and formate dehydrogenase, accumulated and were converted to their active forms upon incubation of cell suspensions with molybdate and chloramphenicol. The conversion to the active enzymes did not occur in cell extracts. When incubated with [(99)Mo]molybdate and chloramphenicol, the tungstate-grown cells incorporated (99)Mo into protein components which were released from membranes by procedures used to release nitrate reductase and formate dehydrogenase and which migrated with these activities on polyacrylamide gels. Although neither activity was formed during incubation of the crude extract with molybdate, (99)Mo was incorporated into protein components which were released from the membrane fraction under the same conditions and were similar to the active enzymes in their electrophoretic properties. The in vitro incorporation of (99)Mo occurred specifically into these components and was equal to or greater than the amount incorporated in vivo under the same conditions. Molybdenum in preformed, active nitrate reductase and formate dehydrogenase did not exchange with [(99)Mo]molybdate, demonstrating that the observed incorporation depended on the demolybdo forms of the enzymes. We conclude that molybdate may be incorporated into the demolybdo forms both in vivo and in vitro; some unknown additional factor or step, required for active enzyme formation, occurs in vivo but not in vitro under the conditions employed.     

chlD gene function in molybdate activation of nitrate reductase.

chlD mutants of Escherichia coli lack active nitrate reductase but form normal levels of this enzyme when the medium is supplemented with 10-3 M molybdate. When chlD mutants were grown in unsupplemented medium and then incubated with molybdate in the presence of chloramphenicol, they formed about 5% the normal level of nitrate reductase. Some chlD mutants or the wild type grown in medium supplemented with tungstate accumulated an inactive protein which was electrophoretically identical to active nitrate reductase. Addition of molybdate to those cells in the presence of chloramphenicol resulted in the formation of fully induced levels of nitrate reductase. Two chlD mutants, including a deletion mutant, failed to accumulate the inactive protein and to form active enzyme under the same conditions. Insertion of 99-Mo into the enzyme protein paralleled activation; 185-W could not be demonstrated to be associated with the accumulated inactive protein. The rates of activation of nitrate reductase at varying molybdate concentrations indicated that the chlD gene product facilitates the activation of nitrate reductase at concentrations of molybdate found in normal growth media. At high concentrations, molybdate circumvented this function in chlD mutants and appeared to activate nitrate reductase by a mass action process. We conclude that the chlD gene plays two distinguishable roles in the formation of nitrate reductase in E. coli. It is involved in the accumulation of fully induced levels of the nitrate reductase protein in the cell membrane and it facilitates the insertion of molybdenum to form the active enzyme.     

Properties of the periplasmic nitrate reductases from Paracoccus pantotrophus and Escherichia coli after growth in tungsten-supplemented media

Paracoccus pantotrophus grown anaerobically under denitrifying conditions expressed similar levels of the periplasmic nitrate reductase (NAP) when cultured in molybdate- or tungstate-containing media. A native PAGE gel stained for nitrate reductase activity revealed that only NapA from molybdate-grown cells displayed readily detectable nitrate reductase activity. Further kinetic analysis showed that the periplasmic fraction from cells grown on molybdate (3 microM) reduced nitrate at a rate of V(max)=3.41+/-0.16 micromol [NO(3)(-)] min(-1) mg(-1) with an affinity for nitrate of K(m)=0.24+/-0.05 mM and was heat-stable up to 50 degrees C. In contrast, the periplasmic fraction obtained from cells cultured in media supplemented with tungstate (100 microM) reduced nitrate at a much slower rate, with much lower affinity (V(max)=0.05+/-0.002 micromol [NO(3)(-)] min(-1) mg(-1) and K(m)=3.91+/-0.45 mM) and was labile during prolonged incubation at >20 degrees C. Nitrate-dependent growth of Escherichia coli strains expressing only nitrate reductase A was inhibited by sub-mM concentrations of tungstate in the medium. In contrast, a strain expressing only NAP was only partially inhibited by 10 mM tungstate. However, none of the above experimental approaches revealed evidence that tungsten could replace molybdenum at the active site of E. coli NapA. The combined data show that tungsten can function at the active site of some, but not all, molybdoenzymes from mesophilic bacteria.     

The role of nitrate reductase in the degradation of the explosive RDX (hexahydro-1,3,5-trinitro-1,3,5-triazine) by <Emphasis Type="Italic">Penicillium</Emphasis> sp. AK96151

In liquid culture on a defined growth medium, Penicillium sp. AK96151 efficiently degraded the explosive hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX, hexogen), causing > 80 % disappearance after 10 d. RDX degradation was reduced to a basal level (< 15 % degraded after 10 d) by the presence of > 150 μM ammonium ions or when the molybdenum component of the medium was replaced by sodium tungstate. An equivalent effect of ammonium, molybdenum and tungsten was observed in protoplasts of this fungus assayed for nitrate reductase activity. This enzyme was not inhibited by RDX itself. The involvement of a nitrate reductase in RDX degradation by Penicillium has practical implications for bioremediation strategies which are discussed.     

Molybdenum and iron as functional constituents of the enzymes of the nitrate-reducing system of Azotobacter chroococcum

The roles of molybdenum and iron in the enzymes of the assimilatory nitrate-reducing system from Azotobacter chroococcum have been investigated.

1. By adding ⁹⁹Mo-molybdate to a cell culture of A. chroococcum with nitrate as the nitrogen source, it has been possible to inccrporate the radioactive metal into a purified preparation of the enzyme nitrare reductase.
2. When ¹⁸⁵W-tungstate was supplied to a culture medium lacking added molybdate, a ¹⁸⁵W-labelled nitrate reductase preparation with negligible activity could be obtained. This in vivo incorporation of tungsten was competitively hindered by molybdenum.
3. The cellular level of nitrite reductase activity gradually increased in response to the addition of increasing amounts of iron to the culture medium. Under the same conditions, the level of nitrate reductase activity was not affected.

     

Isolation,Purification, and Characterization of Nitrate Reductase from a Salt-Tolerant <Emphasis Type="Italic">Rhodotorula glutinis</Emphasis> Yeast Strain Grown in the Presence of Tungsten

The salt-tolerant Rhodotorula glutinis yeast strain grew in medium containing nitrate, 1 mM tungsten, and trace amounts of molybdenum (as impurities from the reagents used). Isolation of electrophoretically homogenous preparation of nitrate reductase from the Rh. glutinis cells grown under these growth conditions is described. The isolated nitrate reductase is a molybdenum-containing homodimer with molecular mass of 130 kD, containing 0.177 mol of Mo per mol of the enzyme. The activity of the enzyme is maximal at pH 7.0 and 35-45 degrees C and is inhibited by low concentrations of azide and cyanide. The enzyme is almost insensitive to 1 mM tungsten.     

Involvement of the narJ and mob gene products in distinct steps in the biosynthesis of the molybdoenzyme nitrate reductase in Escherichia coli

The Escherichia coli mob locus is required for synthesis of active molybdenum cofactor, molybdopterin guanine dinucleotide. The mobB gene is not essential for molybdenum cofactor biosynthesis because a deletion of both mob genes can be fully complemented by just mobA. Inactive nitrate reductase, purified from a mob strain, can be activated in vitro by incubation with protein FA (the mobA gene product), GTP, MgCl₂, and a further protein fraction, factor X. Factor X activity is present in strains that lack MobB, indicating that it is not an essential component of factor X, but over-expression of MobB increases the level of factor X. MobB, therefore, can participate in nitrate reductase activation. The narJ protein is not a component of mature nitrate reductase but narJ mutants cannot express active nitrate reductase A. Extracts from narJ strains are unable to support the in vitro activation of purified mob nitrate reductase: they lack factor X activity. Although the mob gene products are necessary for the biosynthesis of all E. coli molybdoenzymes as a result of their requirement for molybdopterin guanine dinucleotide, NarJ action is specific for nitrate reductase A. The inactive nitrate reductase A derivative in a narJ strain can be activated in vitro following incubation with cell extracts containing the narJ protein. NarJ acts to activate nitrate reductase after molybdenum cofactor biosynthesis is complete.     

Nitrate as a sole nitrogen source forMethanococcus thermolithotrophicus and its effect on growth of several methanogenic bacteria

Methanococcus (Mc.) thermolithotrophicus can use nitrate as the sole source of nitrogen, but four other species of methanogens cannot. The growth rate was similar on both nitrate and ammonium, but yields were 20–25% lower on nitrate.Mc. thermolithotrophicus, Methanobacterium thermoautotrophicum, andMethanobrevibacterium smithii were not inhibited by 20 mM nitrate, butMethanospirillum hungatei was inhibited 35%, andMethanosarcina barkeri was completely inhibited by 20 mM nitrate. WhenMc. thermolithotrophicus was growing with nitrate as the sole source of nitrogen, growth was dependent on either molybdenum or tungsten, and the presence of both gave the best growth response; vanadium or chromium did not replace the requirement for these metals. Growth on ammonium could not be strictly demonstrated to require either of these metals, but both molybdenum and tungsten stimulated growth.     

Biosynthesis of the iron-molybdenum cofactor and the molybdenum cofactor in Klebsiella pneumoniae: effect of sulfur source.

NifQ- and Mol- mutants of Klebsiella pneumoniae show an elevated molybdenum requirement for nitrogen fixation. Substitution of cystine for sulfate as the sulfur source in the medium reduced the molybdenum requirement of these mutants to levels required by the wild type. Cystine also increased the intracellular molybdenum accumulation of NifQ- and Mol- mutants. Cystine did not affect the molybdenum requirement or accumulation in wild-type K. pneumoniae. Sulfate transport and metabolism in K. pneumoniae were repressed by cystine. However, the effect of cystine on the molybdenum requirement could not be explained by an interaction between sulfate and molybdate at the transport level. Cystine increased the molybdenum requirement of Mol- mutants for nitrate reductase activity by at least 100-fold. Cystine had the same effect on the molybdenum requirement for nitrate reductase activity in Escherichia coli ChlD- mutants. This shows that cystine does not have a generalized effect on molybdenum metabolism. Millimolar concentrations of molybdate inhibited nitrogenase and nitrate reductase derepression with sulfate as the sulfur source, but not with cystine. The inhibition was the result of a specific antagonism of sulfate metabolism by molybdate. The effects of nifQ and mol mutations on nitrogenase could be suppressed either by the addition of cystine or by high concentrations of molybdate. This suggests that a sulfur donor and molybdenum interact at an early step in the biosynthesis of the iron-molybdenum cofactor. This interaction might occur nonenzymatically when the levels of the reactants are high.     

Activation of nit-1 nitrate reductase by W-formate dehydrogenase

Formate dehydrogenase ( FDH ) from Clostridium thermoaceticum is a known tungsten enzyme. FDH was tested for the presence of nitrogenase-type cofactor and nitrate reductase-type cofactor by the Azotobacter vinelandii UW-45 and Neurospora crassa nit-1 reconstitution assays, respectively. Tungsten formate dehydrogenase (W- FDH ), containing only a small Mo impurity, activated the nit-1 nitrate reductase extracts when molybdate was also added, but not when tungstate was added. These results show W- FDH contains the cofactor common to all known Mo-enzymes except nitrogenase. The difference between the redox chemistries of W- FDH and W-substituted sulfite oxidase appears to relate to differences in tungsten ligation other than that donated by the cofactor or to variations in the protein environment surrounding the tungsten active site.