酰胺态氮瞬间调节荚膜红假单孢菌光合固氮活性的GS传感机制

天门冬酰胺(Asn)和谷氨酰胺(Gln)对荚膜红假单孢菌固氮酶活性抑制,在表观上类似于氨关闭效应,这种抑制效应由GS参与,相似于氨抑的传感机制。中断Gln代谢的6-diazo-5-oxo-L-norleucine(DON)存在时,氨抑的持续时间延长,与此相类似,Gln抑制加剧,这可能归之于Gln的积累。但是,Gln抑制被methionine sulfoximine(MSX,GS的抑制剂)消除,消除时MSX对Gln的浓度比值约为0.2,与氨抑消除所需的MSX对氨的浓度比值相当。此外,MSX消除氨抑不为DON拮抗,表明Gln抑制固氮酶活性由GS传感。然而,不能抑制GS转谷酰基活性的methionine suffone(MSF,谷氨酸的类似物)却与MSX相同,能消除Gln和氨对固氮活性的抑制。上述观察结果也可延伸至Asn的关闭固氮酶活性效应。     

Inhibition of nitrogenase activity by NH+4 in Rhodospirillum rubrum.

Nitrogenase activities and the patterns of in vivo inhibition of nitrogenase by NH+4 were compared in Rhodospirillum rubrum grown under several conditions of nitrogen availability. In cells grown on N2 or glutamate plus N2, nitrogenase activity was relatively low and was totally inhibited by added NH+4 in 15 to 20 min. In contrast, cells grown on glutamate alone displayed higher nitrogenase activity, and NH+4 had very little effect. Cells grown on limiting amounts of NH+4 had lower nitrogenase activity, but NH+4 produced little inhibitory effect. Uptake of NH+4 could be demonstrated under all of these conditions, and this uptake was blocked by DL-methionine-dl-sulfoximine. The data indicated that cells not recently exposed to NH+4 had no mechanism for rapidly turning off nitrogenase activity in response to sudden additions of NH+4. In contrast, cells grown in the presence of N2, which form NH+4 internally, inhibited nitrogenase activity relatively quickly in response to added NH+4.     

Short-term ammonium inhibition of nitrogen fixation in Azotobacter

Addition of NH4Cl at low concentrations to Azotobacter chroococcum cells caused an immediate cessation of nitrogenase activity, which was recovered once the added NH+4 was exhausted from the medium. In the presence of inhibitors of ammonium assimilation, such as L-methionine-DL-sulfoximine, L-methionine sulfone or 6-diazo-5-oxo-L-norleucine, externally added NH+4 had no effect on nitrogenase activity and the newly-fixed nitrogen was excreted into the medium as NH+4. It is concluded that, in A. chroococcum, NH+4 must be assimilated to exert its short-term inhibitory effect on nitrogen fixation.     

氨和谷氨酰胺对浑球红假单胞菌（Rhodobacter sphaeroides）固氮酶...

浑球红假单胞菌菌株601具有迅速对外源氨作出“关闭”固氮酶活性的反应。氨对固氮酶的抑制作用,可被谷氨酰胺合成酶(GS)抑制剂MSX所解除。反之,加入Glu代谢抑制剂DON,可延长氨抑制的持续时间。Gln对固氮酶也有抑制作用。在脱腺苷化GS的透性细胞中,加入Gln可抑制固氮酶活性,同时,GS腺苷化状态提高。然而,氨则对透性细胞的固氮酶活性和GS腺苷化状态没有影响。     

Regulation of nitrogenase activity by ammonium chloride in Azospirillum spp.

Ammonium chloride (greater than or equal to 0.05 mM) effectively and reversibly inhibited the nitrogenase activity of Azospirillum brasilense, Azospirillum lipoferum and Azospirillum amazonense. The glutamine synthetase inhibitor L-methionine-DL- sulfoximine abolished this "switch-off" in A. lipoferum and A. brasilense, but not in A. amazonense. Azaserine, an inhibitor of glutamate synthase, inhibited nitrogenase activity itself. This provides further evidence for glutamine as a metabolite of regulatory importance in the NH4+ switch-off phenomenon. In A. brasilense and A. lipoferum, a transition period before the complete inhibition of nitrogenase activity after the addition of 1 mM ammonium chloride was observed. The in vitro nitrogenase activity also was decreased after treatment with ammonium. During sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a second dinitrogenase reductase (Fe protein) subunit appeared, which migrated in coincidence with the modified subunit of the inactive Fe protein of the nitrogenase of Rhodospirillum rubrum. After the addition of ammonium 32P was incorporated into this subunit of the Fe protein of A. brasilense. In A. amazonense, the inhibition of nitrogenase activity by ammonium was only partial, and no transition period could be observed. The in vitro nitrogenase activity of ammonium-treated cells was not decreased, and no evidence for a modified Fe protein subunit was found. Nitrogenase extracts of A. amazonense were active and had an Fe protein that migrated as a close double band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Changes in the regulatory form of Rhodospirillum rubrum nitrogenase as influenced by nutritional and environmental factors.

The photosynthetic bacterium Rhodospirillum rubrum regulates the activity of its nitrogenase (N2ase) by interconverting the enzyme into three distinct enzymatic species: N2ase A (a fully active form) and two regulatory forms, N2ase Ractive and N2ase Rinactive. N2ase R is distinguished from N2ase A in vitro by the requirement of its Fe protein for activation by a Mn2+-dependent activating factor. N2ase is converted from the A to the R form in response to certain environmental factors such as carbon starvation, depletion of intracellular adenosine triphosphate, or the addition of NH4+ (or glutamate) to a culture of N-starved cells. The rapid inhibition of R. rubrum N2ase in vivo by NH4+ was shown to result from the conversion of N2ase A to N2ase Rinactive. On depletion of NH4+ from the culture, whole-cell N2ase activity returned; however, the enzyme remained in the R form. Unlike the effect of NH4+, adding glutamate to cells containing N2ase A did not inhibit in vivo activity, but converted the enzyme to the R form (N2ase Ractive). Although glutamate-induced N2ase R formation was much slower than the NH4+-induced reaction, it occurred in the presence of rifampin, indicating that de novo protein synthesis was not involved. This suggested that N2ase R was formed by a modification of N2ase A. Although glutamine synthetase in involved in the conversion of N2ase A to R, the adenylylation state of glutamine synthetase appears not to be involved in regulating this nitrogenase reaction.     

Presence of a second mechanism for the posttranslational regulation of nitrogenase activity in Azospirillum brasilense in response to ammonium.

Although ADP-ribosylation of dinitrogenase reductase plays a significant role in the regulation of nitrogenase activity in Azospirillum brasilense, it is not the only mechanism of that regulation. The replacement of an arginine residue at position 101 in the dinitrogenase reductase eliminated this ADP-ribosylation and revealed another regulatory system. While the constructed mutants had a low nitrogenase activity, NH4+ still partially inhibited their nitrogenase activity, independent of the dinitrogenase reductase ADP-ribosyltransferase/dinitrogenase reductase activating glycohydrolase (DRAT/DRAG) system. These mutated dinitrogenase reductases also were expressed in a Rhodospirillum rubrum strain that lacked its endogenous dinitrogenase reductase, and they supported high nitrogenase activity. These strains neither lost nitrogenase activity nor modified dinitrogenase reductase in response to darkness and NH4+, suggesting that the ADP-ribosylation of dinitrogenase reductase is probably the only mechanism for posttranslational regulation of nitrogenase activity in R. rubrum under these conditions.     

Photoproduction of ammonium ion from N2 in Rhodospirillum rubrum.

NH+4 excretion was undetectable in N2-fixing cultures of Rhodospirillum rubrum (S-1) and nitrogenase activity in these cultures was repressed by the addition of 10 mM NH+4 to the medium. The glutamate analog, L-methionine-DL-sulfoximine (MSX), derepressed N2 fixation even in the presence of 10 mM extracellular NH+4. When 10 mg MSX/ml was added to cultures just prior to nitrogenase induction they developed nitrogenase activity (20% of the control activities) and excreted most of their fixed N2 as NH+4. Nitrogenase activities and NH+4 production from fixed N2 were increased considerably when a combined nitrogen source, NH+4 (greater than 40 mumoles NH+4/mg cell protein in 6 days) or L-glutamate (greater than 60 mumoles NH+4/ mg cell protein in 6 days) was added to the cultures together with MSX. Biochemical analysis revealed that R. rubrum produced glutamine synthetase and glutamate synthase (NADP-dependent) but no detectable NADP-dependent glutamate dehydrogenase. The specific activity of glutamine synthetase was observed to be maximal when nitrogenase activity was also maximal. Nitrogenase and glutamine synthetase activities were repressed by NH+4 as well as by glutamate. The results demonstrate that utilization of solar energy to photoproduce large quantities of NH+4 from N2 is possible with photosynthetic bacteria by interfering with their regulatory control of N2 fixation.     

The glutamine-utilizing site of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase

Reaction of Bacillus subtilis glutamine phosphoribosylpyrophosphate amidotransferase with 6-diazo-5-oxo-L-norleucine resulted in complete loss of its ability to catalyze glutamine-dependent phosphoribosylamine formation and its glutaminase activity, whereas its ability to catalyze ammonia-dependent phosphoribosylamine formation and to hydrolyze phosphoribosylpyrophosphate was increased. The site of reaction with 6-diazo-5-oxo-L-norleucine was the NH2-terminal cysteine residue. The NH2-terminal sequence of the B. subtilis enzyme was homologous with that of the corresponding amidotransferase from Escherichia coli, for which the NH2-terminal cysteine is also essential for glutamine utilization (Tso, J. Y., Hermodson, M. A., and Zalkin, H. (1982) J. Biol. Chem. 257, 3532-3536). The fact that the metal-free E. coli amidotransferase contains a glutamine-utilizing structure that is very similar to that found in B. subtilis amidotransferase, which contains an essential [4Fe-4S] center, indicates that the iron-sulfur center probably plays no role in glutamine utilization.     

Nitrogenase from Rhodospirillum rubrum. Relation between 'switch-off' effect and the membrane component. Hydrogen production and acetylene reduction with different nitrogenase component ratios.

Nitrogenase activity of 'membrane-free' extracts, produced from nitrogen-starved Rhodospirillum rubrum to which 4 mM NH4+ had been added is only about 10% of the activity in the control. The activity could be restored to 80% by including the membrane component, earlier found to activate R. rubrum nitrogenase, in the reaction mixture. The relation between this 'switch-off/switch-on' effect and the function of the membrane component is discussed. Hydrogen production catalyzed by R. rubrum nitrogenase is also dependent on activation by the membrane component. Hydrogen production is inhibited by acetylene but the degree of inhibition is dependent on the nitrogenase component ratio. The strongest inhibition is achieved at low MoFe protein/Fe protein rations. The ATP/2E- values are 4-5 at the component ratios giving the highest activity and increase at high MoFe protein/Fe protein ratios. CO inhibits acetylene reduction but has no effect on the hydrogen production.     

Changes in amino acid and nucleotide pools of Rhodospirillum rubrum during switch-off of nitrogenase activity initiated by NH4+ or darkness.

Amino acid and nucleotide pools were measured in nitrogenase-containing Rhodospirillum rubrum cultures during NH4+- or dark-induced inactivation (switch-off) of the Fe protein. A big increase in the glutamine pool size preceded NH4+ switch-off of nitrogenase activity, but the glutamine pool remained unchanged during dark switch-off. Furthermore, methionine sulfoximine had no effect on the rate of dark switch-off, suggesting that glutamine plays no role in this process. In the absence of NH4+ azaserine, an inhibitor of glutamate synthate, raised glutamine pool levels sufficiently to initiate switch-off in vivo. While added NH4+ substantially increased the size of the nucleotide pools in N-limited cells, the kinetics of nucleotide synthesis were all similar and followed (rather than preceded) Fe protein inactivation. Darkness had little effect on nucleotide pool sizes. Glutamate pool sizes were also found to be important in NH4+ switch-off because of the role of this molecule as a glutamine precursor. Much of the diversity reported in the observations on NH4+ switch-off appears to be due to variations in glutamate pool sizes prior to the NH4+ shock. The nitrogen nutritional background is an important factor in determining whether darkness initiates nitrogenase switch-off; however, no link has yet been established between this and NH4+ (glutamine) switch-off.     

Inactivation of rat renal phosphate-dependent glutaminase with 6-diazo-5-oxo-L-norleucine. Evidence for interaction at the glutamine binding site.

Inactivation of rat renal phosphate-dependent glutaminase by 6-diazo-5-oxo-L-norleucine occurs only under conditions where the enzyme is catalytically active. The glutaminase activity and the rate of inactivation by the diazoketone exhibit very similar phosphate concentration-dependent activation profiles. Because of this phosphate dependency, it was not possible to differentiate an apparent protection by glutamine from the strong inhibition of inactivation caused by glutamate. The ability of glutamate to protect the glutaminase against inactivation is reversed by increasing concentrations of phosphate.The observed characteristics of inactivation by 6-diazo-5-oxo-L-norleucine differ considerably from those reported for the inactivation by L-2-amino-4-oxo-5-chloropentanoic acid. In addition, the presence of o-carbamoyl-L-serine was found to stimulate inactivation by 6-diazo-5-oxo-L-norleucine, but to protect the glutaminase against inactivation by the chloroketone. Preinactivation of the glutaminase by the diazoketone only slightly reduced the stoichiometry of binding of [5-14C]chloroketone. These observations suggest that 6-diazo-5-oxo-L-norleucine and L-2-amino-4-oxo-5-chloropentanoic acid interact with different sites on the glutaminase which are specific for binding glutamine and glutamate, respectively.     

光与氨对Rhodopseudomonas capsulata固氮活性的调节

光强是调节氨瞬间抑制Rps.capsulata光合固氮活性的一个因子。与弱光(500 lx)比较,强光(30000 lx)对固氮活性氨抑的启动推迟。被氨抑制了的固氮活性在强光下较在弱光下提前解抑。经光合作用解联剂处理的菌体,强光拮抗固氮活性氨抑的现象消失。菌体ATP库水平分析表明:在氨关闭固氮活性时,库量升高。氨的同化被阻抑时,氨对光合固氮的瞬间抑制消失,菌体ATP库保持恒定。对光强与氨抑制固氮活性之间可能涉及的机制进行了探讨。     

H2 metabolism in the photosynthetic bacterium Rhodopseudomonas capsulata: production and utilization of H2 by resting cells.

Photoproduction of H2 and activation of H2 for CO2 reduction (photoreduction) by Rhodopseudomonas capsulata are catalyzed by different enzyme systems. Formation of H2 from organic compounds is mediated by nitrogenase and is nto inhibited by an atmosphere of 99% H2. Cells grown photoheterotrophically on C4 dicarboxylic acids (with glutamate as N source) evolve H2 from the C4 acids and also from lactate and pyruvate; cells grown on C3 carbon sources, however, are inactive with the C4 acids, presumably because they lack inducible transport systems. Ammonia is known to inhibit N2 fixation by photosynthetic bacteria, and it also effectively prevents photoproduction of H2; these effects are due to inhibition and, in part, inactivation of nitrogenase. Biosynthesis of the latter, as measured by both H2 production and acetylene reduction assays, is markedly increased when cells are grown at high light intensity; synthesis of the photoreduction system, on the other hand, is not appreciably influenced by light intensity during photoheterotrophic growth. The photoreduction activity of cells grown on lactate + glutamate (which contain active nitrogenase) is greatly activated by NH4+, but this effect is not observed in cells grown with NH4+ as N source (nitrogenase repressed) or in a Nif- mutant that is unable to produce H2. Lactate, malate, and succinate, which are readily used as growth substrates by R. capsulata and are excellent H donors for photoproduction of H2, abolish photoreduction activity. The physiological significances of this phenomenon and of the reciprocal regulatory effects of NH4+ on H2 production and photoreduction are discussed.     

Inhibition of nitrogenase activity by ammonium chloride in Azotobacter vinelandii.

In Azotobacter vinelandii cells, the short-term inhibition of nitrogenase activity by NH4Cl was found to depend on several factors. The first factor is the dissolved oxygen concentration during the assay of nitrogenase. When cells are incubated with low concentrations of oxygen, nitrogenase activity is low and ammonia inhibits strongly. With more oxygen, nitrogenase activity increases. Cells incubated with an optimum amount of oxygen have maximum nitrogenase activity, and the extent of inhibition by ammonia is small. With higher amounts of oxygen, the nitrogenase activity of the cells is decreased and strongly inhibited by ammonia. The second factor found to be important for the inhibition of nitrogenase activity by NH4Cl was the pH of the medium. At a low pH, NH4+ inhibits more strongly than at a higher pH. The third factor that influenced the extent of ammonia inhibition was the respiration rate of the cells. When cells are grown with excess oxygen, the respiration rate of the cells is high and inhibition of nitrogenase activity by ammonia is small. Cells grown under oxygen-limited conditions have a low respiration rate and NH4Cl inhibition of nitrogenase activity is strong. Our results explain the contradictory reports described in the literature for the NH4Cl inhibition of nitrogenase in A. vinelandii.     

Effect of an ntrBC mutation on the posttranslational regulation of nitrogenase activity in Rhodospirillum rubrum.

Homologs of ntrB and ntrC genes from Rhodospirillum rubrum were cloned and sequenced. A mutant lacking ntrBC was constructed, and this mutant has normal nitrogenase activity under nif-derepressing conditions, indicating that ntrBC are not necessary for the expression of the nif genes in R. rubrum. However, the post-translational regulation of nitrogenase activity by ADP-ribosylation in response to NH4+ was partially abolished in this mutant. More surprisingly, the regulation of nitrogenase activity in response to darkness was also affected, suggesting a physiological link between the ntr system and energy signal transduction in R. rubrum. The expression of glutamine synthetase, as well as its posttranslational regulation, was also altered in this ntrBC mutant.     

Regulation of nitrogenase activity by oxygen in Azospirillum brasilense and Azospirillum lipoferum.

The nitrogenase activity of the microaerophilic bacteria Azospirillum brasilense and A. lipoferum was completely inhibited by 2.0 kPa of oxygen (approximately 0.02 atm of O2) in equilibrium with the solution. The activity could be partially recovered at optimal oxygen concentrations of 0.2 kPa. In contrast to the NH4+ switch off, no covalent modification of the nitrogenase reductase (Fe protein) was involved, as demonstrated by Western-blotting and 32P-labeling experiments. However, the inhibition of the nitrogenase activity under anaerobic conditions was correlated with covalent modification of the Fe protein. In contrast to the NH4+ switch off, no increase in the cellular glutamine pool and no modification of the glutamine synthetase occurred under anaerobic switch-off conditions. Therefore, a redox signal, independent of the nitrogen control of the cell, may trigger the covalent modification of the nitrogenase reductase of A. brasilense and A. lipoferum.     

Posttranslational regulatory system for nitrogenase activity in Azospirillum spp.

The mechanism for "NH4+ switch-off/on" of nitrogenase activity in Azospirillum brasilense and A. lipoferum was investigated. A correlation was established between the in vivo regulation of nitrogenase activity by NH4Cl or glutamine and the reversible covalent modification of dinitrogenase reductase. Dinitrogenase reductase ADP-ribosyltransferase (DRAT) activity was detected in extracts of A. brasilense with NAD as the donor molecule. Dinitrogenase reductase-activating glycohydrolase (DRAG) activity was present in extracts of both A. brasilense and A. lipoferum. The DRAG activity in A. lipoferum was membrane associated, and it catalyzed the activation of inactive nitrogenase (by covalent modification of dinitrogenase reductase) from both A. lipoferum and Rhodospirillum rubrum. A region homologous to R. rubrum draT and draG was identified in the genomic DNA of A. brasilense as a 12-kilobase EcoRI fragment and in A. lipoferum as a 7-kilobase EcoRI fragment. It is concluded that a posttranslational regulatory system for nitrogenase activity is present in A. brasilense and A. lipoferum and that it operates via ADP-ribosylation of dinitrogenase reductase as it does in R. rubrum.     

Omega-amidase pathway in the degradation of glutamine in Neurospora crassa.

Evidence for the participation of the glutamine transaminase-omega-amidase pathway in the utilization of glutamine in Neurospora crassa was obtained. Its participation is indicated by the in vitro activities of glutamine transaminase and omega-amidase, the in vivo accumulation of alpha-ketoglutaramate when an inhibitor of transamidases is present, and the inhibition by aminooxyacetic acid and 6-diazo-5-oxo-L-norleucine of the ammonium excreted in the presence of glutamine by a mutant strain that lacks glutamate dehydrogenase and glutamate synthase.     

The inhibition by 6-diazo-5-oxo-l-norleucine of glutamine catabolism of the cultured human lymphoblast.

The rapid catabolism of glutamine by the cultured human lymphoblast line WI-L2 can be inhibited greater than 95% by incubation of cell suspensions with 6-diazo-5-oxo-L-norleucine (DON). The inhibition persists for at least four hours after removal of DON from the cell suspension. The exposure of cells to DON ihibits over 95% of the glutaminase activity measured in lysates in the presence of either phosphate or maleate. Similarly, gamma-glutamyl transpeptidase, assayed with gamma-glutamyl-p-nitroanilide as substrate and glycyglycine as acceptor, is inhibited over 90%. DON-treated and control cells accumulated radioactive material from suspensions containing [14C]-L-glutamine at similar initial rates; the radioactive material accumulated by the DON-treated cells is all recoverable as glutamine while the radioactive material accumulated by untreated cells is principally recovered as glutamate.