Properties and regulation of glutamine synthetase from Rhodospirillum rubrum.

Glutamine synthetase from Rhodospirillum rubrum was purified and characterized with respect to its pH optimum and the effect of Mg2+ on its active and inactive forms. Both adenine and phosphorus were incorporated into the inactive form of the enzyme, indicating covalent modification by AMP. The modification could not be removed by phosphodiesterase. Evidence for regulation of the enzyme by oxidation was obtained. Extracts from oxygen-treated cells had lower specific activities than did extracts from cells treated anaerobically. Glutamine synthetase activity was found to decrease in the dark in phototrophically grown cells; activity was recovered on re-illumination.     

Purification and partial characterization of glutamine synthetase from the photosynthetic bacterium Rhodospirillum rubrum

Glutamine synthetase (L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2) from the photosynthetic bacterium Rhodospirillum rubrum grown under nitrogen fixing conditions has been purified to homogeneity. The purification procedure involves affinity chromatography on ADP-agarose type 2 as the major purification step. The recovery in the purification is 70%. The specific activity of the purified enzyme is about 10-times higher in the gamma-glutamyl transferase assay than in the coupled biosynthetic assay. The molecular weight was determined to 530,000 by native gradient polyacrylamide gel electrophoresis and to 500,000 by gel filtration. The subunits have an apparent molecular weight of 52,000. Glutamine synthetase isolated from Rsp. rubrum which had been exposed to ammonium ions ('switch-off') before harvest had about 20% of the transferase activity compared with the enzyme purified from nitrogen-starved cells. The low-activity form showed two bands on sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

NAD-dependent cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase from Rhodospirillum rubrum.

Chemical cross-linking of dinitrogenase reductase and dinitrogenase reductase ADP-ribosyltransferase (DRAT) from Rhodospirillum rubrum has been investigated with a cross-linking system utilizing two reagents, 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide and sulfo-N-hydroxysuccinimide. Cross-linking between dinitrogenase reductase and DRAT requires the presence of NAD, the cellular ADP-ribose donor, or a NAD analog containing an unmodified nicotinamide group, such as nicotinamide hypoxanthine dinucleotide. NADP, which will not replace NAD in the modification reaction, does support cross-linking between dinitrogenase reductase and DRAT. The DRAT-catalyzed ADP-ribosylation of dinitrogenase reductase is inhibited by sodium chloride, as is the cross-linking between dinitrogenase reductase and DRAT, suggesting that ionic interactions are required for the association of these two proteins. Cross-linking is specific for native, unmodified dinitrogenase reductase, in that both oxygen-denatured and ADP-ribosylated dinitrogenase reductase fail to form a cross-linked complex with DRAT. The ADP-bound and adenine nucleotide-free states of dinitrogenase reductase form cross-linked complexes with DRAT; however, cross-linking is inhibited when dinitrogenase reductase is in its ATP-bound state.     

Low- and high-activity forms of glutamine synthetase from Rhodospirillum rubrum: sensitivity to feed-back effectors and activation of the low-activity form.

Glutamine synthetase from Rhodospirillum rubrum can be isolated in two forms, with low and high activity, respectively, depending on the concentration of combined nitrogen in the medium before harvest. The two forms have been studied with respect to their dependence on Mn2+ and Mg2+ in both the transferase and the biosynthetic assay. There is no difference in pH optimum between the forms in the biosynthetic assay. In addition the pH-optima for the two cations studied are very close, 7.4 (Mg2+) and 7.2 (Mn2+). It also shows that the activity of the low-activity form is higher than that of the high-activity form in the Mn(2+)-dependent biosynthetic assay. The two forms of Rsp. rubrum glutamine synthetase have also been studied with respect to their sensitivity towards feed-back effectors. In the transferase assay both forms are inhibited to essentially the same degree by alanine, glycine, histidine, AMP, CTP and UTP, CTP being the most effective of the nucleotides and of the amino acids alanine causes the highest inhibition. In the biosynthetic assay these effectors show different degrees of inhibition on the two different forms; the high-activity form being the most sensitive. The results are discussed in relation to properties of glutamine synthetase from Escherichia coli and other phototropic bacteria in which regulation of glutamine synthetase is known to be due to adenylylation. It is also shown that the low-activity form of Rsp. rubrum glutamine synthetase can be activated in crude extracts in a reaction that is inhibited by glutamine.     

The poor growth of Rhodospirillum rubrum mutants lacking PII proteins is due to an excess of glutamine synthetase activity

The P(II) family of proteins is found in all three domains of life and serves as a central regulator of the function of proteins involved in nitrogen metabolism, reflecting the nitrogen and carbon balance in the cell. The genetic elimination of the genes encoding these proteins typically leads to severe growth problems, but the basis of this effect has been unknown except with Escherichia coli. We have analysed a number of the suppressor mutations that correct such growth problems in Rhodospirillum rubrum mutants lacking P(II) proteins. These suppressors map to nifR3, ntrB, ntrC, amtB(1) and the glnA region and all have the common property of decreasing total activity of glutamine synthetase (GS). We also show that GS activity is very high in the poorly growing parental strains lacking P(II) proteins. Consistent with this, overexpression of GS in glnE mutants (lacking adenylyltransferase activity) also causes poor growth. All of these results strongly imply that elevated GS activity is the causative basis for the poor growth seen in R. rubrum mutants lacking P(II) and presumably in mutants of some other organisms with similar genotypes. The result underscores the importance of proper regulation of GS activity for cell growth.     

Glutathione reductase from Rhodospirillum rubrum

Summary Glutathione reductase (NADPH¹: glutathione oxidoreductase (EC 1.6.4.2) was purified 70 fold from Rhodospirillum rubrum by ammonium sulfate fractionation, gelfiltration with Sephadex and chromatography on DEAE-cellulose. The optimum pH of the reaction is 7.5–8.2 K _m values of 8.4×10^–6 M for NADPH and 5.8×10^–5 M for GSSG were determined. The kinetic data indicate a bisubstrate reaction mechanism. The prosthetic group is FAD (K _m 1.1×10^–6M). The flavin can be completely dissociated from the enzyme, and 70% of the original activity can subsequently be restored by FAD. The molecular weight was determined with a calibrated column Sephadex G-200 and found to be approximately 63,000. The enzyme is inhibited reversibly by several anions. With iodide the inhibition is competitive with respect to GSSG. Sulfhydryl reagents (N-ethylmaleinimide, p-chlormercuribenzoate) strongly inhibit the enzyme when it is present in the reduced state. The enzyme is reduced by low concentrations of NADPH and by higher concentrations of NADH. GSSG protects the enzyme against this inhibition. The enzyme is reversibly inhibited by incubation with NADPH or NADH.

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Zusammenfassung Glutathionreduktase wurde aus Rhodospirillum rubrum mit Ammoniumsulfatfraktionierung, Gelfiltration mit Sephadex und Chromatographie an DEAE-Cellulose 70 fach angereichert. Das pH Optimum der Reaktion liegt bei 7,5–8,2. K _m -Werte: 8,4·10^–6 M für NADPH und 5,8·10^–5 M für GSSG. Aus den kinetischen Daten ergibt sich für das Enzym ein Bisubstratreaktionsmechanismus. Die prosthetische Gruppe ist FAD (K _m 1,1·10^–6 M). Das Flavin kann vollständig vom Enzymprotein abdissoziiert werden, durch erneute Zugabe von FAD können etwa 70% der ursprünglichen Aktivität zurückerhalten werden. Das Molekulargewicht, bestimmt durch Gelfiltration mit einer kalibrierten Säule Sephadex G-200, ist ca. 63000. Das Enzym wird durch verschiedene Anionen reversibel gehemmt. Bei J^– ist die Hemmung kompetitiv mit GSSG. Sulfhydrylreagentien (N-Äthylmaleinimid und p-Chlomercuribenzoat) sind potente Inhibitoren, wenn das Enzym im reduzierten Zustand vorliegt. Das Enzym kann bereits durch niedrige Konzentrationen an NADPH sowie durch höhere Konzentrationen an NADH reduziert werden. GSSG schützt das Enzymprotein gegen die Hemmung durch Sulfhydryl-reagentien. Das Enzym wird durch Inkubation mit NADPH und NADH reversibel gehemmt.

     

Carbon monoxide dehydrogenase from Rhodospirillum rubrum

The carbon monoxide dehydrogenase from the photosynthetic bacterium Rhodospirillum rubrum was purified over 600-fold by DEAE-cellulose chromatography, heat treatment, hydroxylapatite chromatography, and preparative scale gel electrophoresis. In vitro, this enzyme catalyzed a two-electron oxidation of CO to form CO2 as the product. The reaction was dependent on the addition of an electron acceptor. The enzyme was oxygen labile, heat stable, and resistant to tryptic and chymotryptic digestion. Optimum in vitro activity occurred at pH 10.0. A sensitive, hemoglobin-based assay for measuring dissolved CO levels is presented. The in vitro Km for CO was determined to be 110 microM. CO, through an unknown mechanism, stimulated hydrogen evolution in whole cells, suggesting the presence of a reversible hydrogenase in R. rubrum which is CO insensitive in vivo.     

o-Phosphotyrosyl glutamine synthetase: modification of the nucleotide ligation site of adenylylated glutamine synthetase

The nucleotide ligation site of adenylylated glutamine synthetase, which contains a unique tyrosyl residue linked through a phosphodiester bond to 5'-AMP, was studied by digestion with three hydrolytic enzymes. The products on micrococcal nuclease digestion were adenosine and o-phosphotyrosyl glutamine synthetase. The Km for this macromolecular substrate with the nuclease was 40 microM, at pH 8.9. The glutamine synthetase activity was not affected by deadenosylation with the nuclease, in contrast to SVPDE digestion, with which the glutamine synthetase activity was markedly increased. The Km for the native adenylylated glutamine synthetase with the SVPDE was 36 microM, i.e., similar to that for the nuclease. When the isolated o-phosphotyrosyl enzyme was incubated with alkaline phosphatase at pH 7.2, the glutamine synthetase activity rapidly increased to the same level as that of the SVPDE treated enzyme. Furthermore, kinetic properties of the o-phosphotyrosyl glutamine synthetase were compared with those of the adenylylated enzyme. The optimum pH, apparent Km for each of three substrates, glutamate, ATP, and NH3, and Vmax were in good agreement, as to either Mg2+- or Mn2+-dependent biosynthetic activity. From these results we can conclude that the regulation of glutamine synthetase activity simply requires the phosphorylation of the tyrosyl residue in each subunit, without recourse to adenylylation.     

A carotenoprotein from chromatophoreses of Rhodospirillum rubrum

A carotenoprotein has been obtained by SDS-solubilization of $\text{Rhodospirillum rubrum}$ chromatophores. It was then purified by (NH₄)₂SO₄ precipitation and Sephadex G-200 filtration. SDS-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of about 12,000. The absorption spectrum of the complex is entirely different from the usual three peaked carotenoid spectrum, it has only a major peak at 370 nm. However, after acetone extraction the spectrum of spirilloxanthin reappears. The fact that the carotenoid associates with a specific protein provides strong evidence that the complex originates from the chromatophores and is not a preparative artefact.     

NAD-, NMN-, and NADP-dependent modification of dinitrogenase reductases from Rhodospirillum rubrum and Azotobacter vinelandii

Nitrogenase activity in the photosynthetic bacterium Rhodospirillum rubrum is reversibly regulated by ADP-ribosylation of a specific arginine residue of dinitrogenase reductase based on the cellular nitrogen or energy status. In this paper, we have investigated the ability of nicotinamide adenine dinucleotide, NAD (the physiological ADP-ribose donor), and its analogs to support covalent modification of dinitrogenase reductase in vitro. R. rubrum dinitrogenase reductase can be modified by DRAT in the presence of 2 mM NAD, but not with 2 mM nicotinamide mononucleotide (NMN) or nicotinamide adenine dinucleotide phosphate (NADP). We also found that the apo- and the all-ferrous forms of R. rubrum dinitrogenase reductase are not substrates for covalent modification. In contrast, Azotobacter vinelandii dinitrogenase reductase can be modified by the dinitrogenase reductase ADP-ribosyl transferase (DRAT) in vitro in the presence of either 2 mM NAD, NMN or NADP as nucleotide donors. We found that: (1) a simple ribose sugar in the modification site of the A. vinelandii dinitrogenase reductase is sufficient to inactivate the enzyme, (2) phosphoADP-ribose is the modifying unit in the NADP-modified enzyme, and (3) the NMN-modified enzyme carries two ribose-phosphate units in one modification site. This is the first report of NADP- or NMN-dependent modification of a target protein by an ADP-ribosyl transferase.     

Isolation and properties of succinate dehydrogenase from Rhodospirillum rubrum

Succinate dehydrogenase has been solubilized from R. rubrum chromatophores with the use of chaotropic agents, and purified approximately 80-fold. The preparation (SD_r) contains 8 g-atoms of iron per mole of flavin, and has a turnover number of approximately 4000 (moles succinate oxidized by ferricyanide or phenazine methosulfate/mole of flavin/min at 38 °C). Its absorption and EPR spectra are similar to those of bovine heart succinate dehydrogenase. SD_r can cross-interact with the bovine heart electron-transport system (alkali-inactivated ETP) and reconstitute succinoxidase activity with an efficiency comparable to the reconstitution activity of purified bovine heart succinate dehydrogenase. Preliminary results suggest that SD_r has a molecular weight of approximately 85,000, and that it is composed of a flavoprotein subunit with a molecular weight of approximately 60,000, plus a second subunit (possibly an iron-sulfur protein) with a molecular weight of approximately 25,000.     

Reversible regulation of the nitrogenase iron protein from Rhodospirillum rubrum by ADP-ribosylation in vitro.

Nitrogenase activity in the photosynthetic bacterium Rhodospirillum rubrum is reversibly regulated by interconversion of the Fe protein between a modified and an unmodified form. Since the discovery of the activation process in 1976, investigators have been unable to demonstrate the inactivation (modification) reaction in vitro. In this study, NAD-dependent modification and concomitant inactivation of the Fe protein were demonstrated in crude extracts of R. rubrum. Activation of the in vitro-modified Fe protein by activating enzyme and structural similarity between the in vivo and in vitro modifications are presented as evidence that the in vitro modification is the physiologically relevant ADP-ribosylation reaction. Using a partially purified preparation, we showed that the inactivating enzyme activity is stimulated by divalent metal ions and ADP, that O2-denatured Fe protein will not serve as a substrate, and that dithionite inhibits the modification reaction.