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Volume 139, Issue 12

Molecular and kinetic characterization of glutamate synthase from the phototrophic bacterium ElFl Free

Abstract

Glutamate synthase (GOGAT) from the phototrophic non-sulphur purple bacterium ElFl has been purified to electrophoretic homogeneity by affinity chromatography. The native protein consisted of two different subunits of 175 and 53 kDa and contained 4 mol FAD, 4 mol iron and 4 mol labile sulphide per mol of dimer enzyme. The enzyme used NADPH as the electron donor and was inhibited by iron-chelating and thiol group reagents. GOGAT exhibited NAD(P)H-diaphorase activity which used sodium ferricyanide, cytochrome and dichlorophenol indophenol as alternative electron acceptors. By contrast, glutaminase activity was not detected in purified GOGAT. The amino acid composition was quite different from that of other bacterial GOGATs, and the protein presented different aggregation states depending on the ionic strength. Two major multimeric active species with Stokes’ radii of 6·18 and 7·32 nm could be separated by gel-filtration of protein solutions made in 0·5 -KCl, whereas in the absence of salt, the maximal GOGAT activity corresponded to an oligomer with Stokes radius of 6·80 nm. The enzyme exhibited apparent negative cooperativity for glutamine, and was competitively inhibited by -glutamate and NADP.

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© Society for General Microbiology, 1993
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1993-12-01
2023-03-22
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References

  1. Adachi K., Suzuki I. 1977; Purification and properties of glutamate synthase from Thiobacillus thioparus. Journal of Bacteriology 129:1173–1182
     [Google Scholar]
  2. Alef K., Zumft W.G. 1981; Regulatory properties of glutamine synthetase from the nitrogen-fixing phototrophic bacterium Rhodo-pseudomonas palustris. Zeitschrift für Naturforschung 36:784–789
     [Google Scholar]
  3. Avila C., Botella J.R., Cánovas F.M., Núñez De Castro I., Valpuesta V. 1987; Different characteristics of the two glutamate synthases in the green leaves of Lycopersicon esculentum. Plant Physiology 85:1036–1039
     [Google Scholar]
  4. Beinert H. 1983; Semi-micro methods for analysis of labile sulfide and of labile sulfide plus sulfane sulfur in unusually stable iron-sulfur proteins. Analytical Biochemistry 131:373–378
     [Google Scholar]
  5. Bradford M.M. 1976; A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Biochemistry 72:248–254
     [Google Scholar]
  6. Brewer J.M., Pesce A.J., Spencer T.E. 1974; Ultracentrifugation. In Experimental Techniques in Biochemistry pp. 161–215 Brewer J.M., Pesce A.J., Ashworth R.B. Edited by Englewood Cliffs, NJ: Prentice-Hall.;
     [Google Scholar]
  7. Caballero F.J., Cejudo F.J., Florencio F.J., Cárdenas J., Castillo F. 1985; Molecular and regulatory properties of glutamine synthetase from the phototrophic bacterium Rhodo-pseudomonas capsulata E1F1. Journal of Bacteriology 162:804–809
     [Google Scholar]
  8. Caballero F.J., Cárdenas J., Castillo F. 1987; Involvement of sulphydryl groups in glutamine synthetase activity from Rhodobacter capsulatus E1F1. FEMS Microbiology Letters 41:7–11
     [Google Scholar]
  9. Caballero F. J., Cárdenas J., Castillo F. 1989a; Purification and properties of l-alanine dehydrogenase of the phototrophic bacterium Rhodobacter capsulatus E1F1. Journal of Bacteriology 171:3205–3210
     [Google Scholar]
  10. Caballero F. J., Igeño I., Cárdenas J., Castillo F. 1989b; Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1. Archives of Microbiology 152:508–511
     [Google Scholar]
  11. Carlberg I., Nordlund S. 1991; Purification and partial characterization of glutamate synthase from Rhodospirillum rubrum grown under nitrogen-fixing conditions. Biochemical Journal 279:151–154
     [Google Scholar]
  12. Castaño I., Bastarrachea F., Covarrubias A.A. 1988; gltBDF operon of Escherichia coli. Journal of Bacteriology 170:821–827
     [Google Scholar]
  13. Castillo F., Moreno-Vivián C., Igeño M.I., Caballero F.J. 1991; Catabolismo de aminoácidos en Rhodobacter capsulatus E1F1. In I Reunion de Biología Molecular de Plantas pp. 161–165 Nicolás G., Sabater B. Edited by Salamanca, Spain: Publicaciones de la Universidad de Salamanca.;
     [Google Scholar]
  14. Dahlquist F.W. 1978; The meaning of Scatchard and Hill plots. Methods in Enzymology 48:270–299
     [Google Scholar]
  15. Doeg K.A., Ziegler D.M. 1962; Simplified methods for the estimation of iron in mitochondria and submitochondrial fractions. Archives of Biochemistry and Biophysics 97:37–40
     [Google Scholar]
  16. Ertan H. 1992; Some properties of glutamate dehydrogenase, glutamine synthetase and glutamate synthase from Corynebacterium callunae. Archives of Microbiology 158:35–41
     [Google Scholar]
  17. Galván F., Márquez A.J., Vega J.M. 1984; Purification and molecular properties of ferredoxin-glutamate synthase from Chlamydomonas reinhardii. Planta 162:180–187
     [Google Scholar]
  18. Gosset G., Merino E., Recillas F., Oliver G., Becerril B., Bolivar F. 1989; Amino acid sequence analysis of the glutamate synthase enzyme from Escherichia coli K-12. Protein Sequences & Data Analysis 2:9–16
     [Google Scholar]
  19. Hemmilä I.A., Mäntsälä P.I. 1978; Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium. Biochemical Journal 173:45–52
     [Google Scholar]
  20. Herbert R.A., Siefert E., Pfennig N. 1978; Nitrogen assimilation in Rhodopseudomonas acidophila. Archives of Microbiology 119:1–5
     [Google Scholar]
  21. Johansson B.C., Gest H. 1976; Inorganic nitrogen assimilation by the photosynthetic bacterium Rhodopseudomonas capsulata. Journal of Bacteriology 128:683–688
     [Google Scholar]
  22. Laemmli U.K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature; London: 227680–685
     [Google Scholar]
  23. Lea P.J., Miflin B.J. 1975; The occurrence of glutamate synthase in algae. Biochemical and Biophysical Research Communications 64:856–862
     [Google Scholar]
  24. Madigan M., Cox S.S. 1982; Nitrogen metabolism in Rhodo-pseudomonas globiformis. Archives of Microbiology 133:6–10
     [Google Scholar]
  25. Madkour M.A., Smith L.T., Smith G.M. 1990; Preferential osmolyte accumulation: a mechanism of osmotic stress adaptation in diazotrophic bacteria. Applied and Environmental Microbiology 56:2876–2881
     [Google Scholar]
  26. Mäntsälä P., Zalkin H. 1976a; Active subunits of Escherichia coli glutamate synthase. Journal of Bacteriology 126:539–541
     [Google Scholar]
  27. Mäntsälä P., Zalkin H. 1976b; Glutamate synthase: properties of the glutamine-dependent activity. Journal of Biological Chemistry 251:3294–3299
     [Google Scholar]
  28. Márquez A.J., Galván F., Vega J.M. 1984; Purification and characterization of the NADH-glutamate synthase from Chlamy-domonas reinhardii. Plant Science Letters 34:305–314
     [Google Scholar]
  29. Martin R.G., Ames B.N. 1961; A method of determining the sedimentation behavior of enzymes: application to protein mixtures. Journal of Biological Chemistry 236:1372–1379
     [Google Scholar]
  30. Matsuoka K., Kimura K. 1986; Glutamate synthase from Bacillus subtilis PCI 219. Journal of Biochemistry 99:1087–1100
     [Google Scholar]
  31. Mei B., Jiao R. 1988; Purification and properties of glutamate synthase from Nocardia mediterranei. Journal of Bacteriology 170:1940–1944
     [Google Scholar]
  32. Miflin B.J., Lea P.J. 1976; The pathway of nitrogen assimilation in plants. Phytochemistry 15:873–885
     [Google Scholar]
  33. Miller R.E., Stadtman E.R. 1972; Glutamate synthase from Escherichia coli. Iron sulfide flavoprotein. Journal of Biological Chemistry 247:7407–7419
     [Google Scholar]
  34. Moreno-Vivián C., Cejudo F.J., Cárdenas J., Castillo F. 1983; Ammonia assimilation pathways in Rhodopseudomonas capsulata E1F1. Archives of Microbiology 136:147–151
     [Google Scholar]
  35. Moreno-Vivián C., Caballero F.J., Martínez-Luque M., Cárdenas J., Castillo F. 1990; Regulation of inorganic nitrogen metabolism in the phototrophic bacterium Rhodobacter capsulatus E1F1. In Inorganic Nitrogen in Plants and Microorganisms pp. 145–150 Ullrich W.R., Rigano C., Fuggi A., Aparicio P.J. Edited by Berlin: Springer-Verlag.;
     [Google Scholar]
  36. Nordlund S., Kanemoto R.H., Murell S.A., Ludden P.W. 1985; Properties and regulation of glutamine synthetase from Rhodospirillum rubrum. Journal of Bacteriology 161:13–17
     [Google Scholar]
  37. Oliver G., Gosset G., Sánchez-Pescador R., Lozoya E., Ku L.M., Flores N., Becerril B., Valle F., Bolivar F. 1987; Determination of the nucleotide sequence for the glutamate synthase structural genes of Escherichia coli K-12. Gene 60:1–11
     [Google Scholar]
  38. Pundak S., Eisenberg M. 1981; Structure and activity of malate dehydrogenase from the extreme halophilic bacteria of the Dead Sea. European Journal of Biochemistry 118:463–470
     [Google Scholar]
  39. Ratti S., Curti B., Zanetti G., Galli E. 1985; Purification and characterization of glutamate synthase from Azospirillum brasilense. Journal of Bacteriology 163:724–729
     [Google Scholar]
  40. Ratti S., Vanoni M.A., Grandori R., Zanetti G., Curti B. 1987; Kinetic studies on glutamate synthase from Azospirillum brasilense. In Flavins and Flavoproteins 1987 pp. 385–389 Edmondson D.E., McCormick D.B. Edited by Berlin: Walter de Gruyter.;
     [Google Scholar]
  41. Romero F., Caballero F.J., Castillo F., Roldán J.M. 1985; Immunoelectrophoretic approach to the metabolic regulation of glutamine synthetase in Rhodopseudomonas capsulata E1F1: role of glutamine. Archives of Microbiology 143:111–116
     [Google Scholar]
  42. Schreier H.J., Bernlohr R.W. 1984; Purification and properties of glutamate synthase from Bacillus licheniformis. Journal of Bacteriology 160:591–599
     [Google Scholar]
  43. Siegel L.M., Monty K.J. 1966; Determination of molecular weights and frictional ratios of proteins in impure systems by use of gE1F1ltration and density gradient centrifugation. Application to crude preparations of sulfite and hydroxylamine reductase. Biochimica et Biophysica Acta 112:346–362
     [Google Scholar]
  44. Singhal R.K., Krishnan I.S., Dua R.D. 1989; Stabilization, purification, and characterization of glutamate synthase from Clostridium pasteurianum. Biochemistry 28:7928–7935
     [Google Scholar]
  45. Smith P.K., Krohn R.I., Hermanson G.T., Mallia A.K., Gartner F.H., Provenzano M.D., Fujimoto E.K., Goeke N.M., Olson B.J., Klenk D.C. 1985; Measurement of protein using bicinchoninic acid. Analytical Biochemistry 150:76–85
     [Google Scholar]
  46. Stewart G, Mann A.F., Fentem P.A. 1980; Glutamate synthase. In The Biochemistry of Plants 5 pp. 309–327 Miflin B.J. Edited by New York: Academic Press.;
     [Google Scholar]
  47. Sung H.C., Tachiki T., Kumagai H., Tochikura T. 1984; Properties of glutamate synthase from Brevibacterium flavum. Journal of Fermentation Technology 62:569–575
     [Google Scholar]
  48. Tempest D.W., Meers J.L., Brown C.M. 1970; Synthesis of glutamate in Aerobacter aerogenes by a hitherto unknown route. Biochemical Journal 117:405–407
     [Google Scholar]
  49. Trotta P.P., Platzer K.E.B., Haschemeyer R.H., Meister A. 1974; Glutamine-binding subunit of glutamate synthase and partial reactions catalyzed by this glutamine amidotransferase. Proceedings of the National Academy of Sciences of the United States of America 714607–4611
     [Google Scholar]
  50. Vanoni M.A., Negri A., Zanetti G., Ronchi S., Curti B. 1990; Structural studies on the subunits of glutamate synthase from Azospirillum brasilense. Biochimica et Biophysica Acta 1039:374–377
     [Google Scholar]
  51. Vanoni M.A., Edmondson D.E., Rescigno M., Zanetti G., Curti B. 1991a; Mechanistic studies on Azospirillum brasilense glutamate synthase. Biochemistry 30:11478–11484
     [Google Scholar]
  52. Vanoni M.A., Nuzzi L., Rescigno M., Zanetti G., Curti B. 1991b; The kinetic mechanism of the reactions catalyzed by the glutamate synthetase from Azospirillum brasilense. European Journal of Biochemistry 202:181–189
     [Google Scholar]
  53. Vanoni M.A., Edmondson D.E., Zanetti G., Curti B. 1992; Characterization of the flavins and the iron-sulfur centers of glutamate synthase from Azospirillum brasilense by absorption, circular dichroism, and electron paramagnetic resonance spectroscopies. Biochemistry 31:4613–4623
     [Google Scholar]
  54. Wang C.C., Raper I.R. 1970; Isozyme patterns and sexual morphogenesis in Schizophilum. Proceedings of the National Academy of Sciences of the United States of America 66882–889
     [Google Scholar]
  55. Woese C.R. 1987; Bacterial evolution. Microbiological Reviews 51:221–271
     [Google Scholar]
  56. Yelton M.M., Yoch D.C. 1981; Nitrogen metabolism in Rhodospirillum rubrum : characterization of glutamate synthase. Journal of General Microbiology 123:335–342
     [Google Scholar]
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Molecular and kinetic characterization of glutamate synthase from the phototrophic bacterium Rhodobacter capsulatus ElFl
Microbiology 139, 2921 (1993); https://doi.org/10.1099/00221287-139-12-2921
/content/journal/micro/10.1099/00221287-139-12-2921
/content/journal/micro/10.1099/00221287-139-12-2921
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