1887

Abstract

Purified glutamate synthase (GOGAT) from was characterized as a high-molecular-mass polypeptide, a distinction shared with previously described GOGATs from other eukaryotic micro-organisms. Using degenerate deoxyoligonucleotides, designed from conserved regions of the alfalfa, maize and GOGAT genes, a 300 bp PCR fragment from the GOGAT gene was obtained. This fragment was used to construct null GOGAT mutants of by gene replacement. These mutants showed no growth defect phenotype and were able to grow on ammonium as sole nitrogen source. Double mutants obtained from a cross between a previously described mutant and the null GOGAT strain were full glutamate auxotrophs. These results indicate that glutamate biosynthesis in . is afforded through the combined action of and products.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-146-1-239
2000-01-01
2024-03-29
Loading full text...

Full text loading...

/deliver/fulltext/micro/146/1/1460239a.html?itemId=/content/journal/micro/10.1099/00221287-146-1-239&mimeType=html&fmt=ahah

References

  1. Adachi K., Suzuki I. 1977; Purification and properties of glutamate synthase from Thiobacillus thioparus. J Bacteriol 129:1173–1182
    [Google Scholar]
  2. Anderson M. P., Vance C. P., Heichel G. H., Miller S. S. 1989; Purification and characterization of NADH-glutamate synthase from alfalfa root nodules. Plant Physiol 90:351–358 [CrossRef]
    [Google Scholar]
  3. Avendaño A., DeLuna A., Olivera H., Valenzuela L., González A. 1997; GDH3 encodes a glutamate dehydrogenase isozyme, a previously unrecognized route for glutamate biosynthesis in Saccharomyces cerevisiae. J Bacteriol 179:5594–5597
    [Google Scholar]
  4. Barel I., MacDonald D. W. 1993; Enzyme defects in glutamate-requiring strains of Schizosaccharomyces pombe. FEMS Microbiol Lett 113:267–272 [CrossRef]
    [Google Scholar]
  5. Benny A. G., Boland M. J. 1977; Enzymes of nitrogen metabolism in legume nodules. Purification and properties of NADH-dependent glutamate synthase from lupine nodules. Eur J Biochem 79:355–362 [CrossRef]
    [Google Scholar]
  6. Bravo A., Mora J. 1988; Ammonium assimilation in Rhizobium phaseoli by the glutamine synthetase-glutamate synthase pathway. J Bacteriol 170:980–984
    [Google Scholar]
  7. Carlberg I., Nordlund S. 1991; Purification and partial characterization of glutamate synthase from Rhodospirillum rubrum grown under nitrogen-fixing conditions. Biochem J 279:151–154
    [Google Scholar]
  8. Chen F. L., Cullimore J. V. 1988; Two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L. Plant Physiol 88:1411–1417 [CrossRef]
    [Google Scholar]
  9. Cogoni C., Valenzuela L., González-Halphen D., Olivera H., Macino G., Ballario P., González A. 1995; Saccharomyces cerevisiae has a single glutamate synthase gene coding for a plant-like high-molecular-weight polypeptide. J Bacteriol 177:792–798
    [Google Scholar]
  10. Doherty D. 1970; l-Glutamate dehydrogenases (yeast). Methods Enzymol 17:850–856
    [Google Scholar]
  11. Gietz R. D., Woods R. A. 1994; High efficiency transformation with lithium acetate. Molecular Genetics of Yeast: a Practical Approach121–134 Oxford: Oxford University Press;
    [Google Scholar]
  12. González A., Membrillo-Hernández J., Olivera H., Aranda C., Macino G., Ballario P. 1992; Cloning of a yeast gene coding for the glutamate synthase small subunit (GUS2) by complementation of Saccharomyces cerevisiae and Escherichia coli glutamate auxotrophs. Mol Microbiol 6:301–308 [CrossRef]
    [Google Scholar]
  13. González-Halphen D., Lindorfer M. A., Capaldi R. A. 1988; Subunit arrangement in beef heart complex III. Biochemistry 27:7021–7031 [CrossRef]
    [Google Scholar]
  14. Hayakawa T., Yamaya T., Kamashi K., Ojima K. 1992; Purification, characterization and immunological properties of NADH-dependent glutamate synthase from rice cell cultures. Plant Physiol 98:1317–1322 [CrossRef]
    [Google Scholar]
  15. Helling R. B. 1994; Why does Escherichia coli have two primary pathways for synthesis of glutamate?. J Bacteriol 176:4664–4668
    [Google Scholar]
  16. Holmes A. R., Collings A., Farnden K. J. F., Sheperd M. G. 1989; Ammonium assimilation by Candida albicans and other yeasts: evidence for activity of glutamate synthase. J Gen Microbiol 135:1423–1430
    [Google Scholar]
  17. Holzer H., Schneider S. 1957; Anreicherung und Trennung einer DPN-spezifischen und einer TPN-spezifischen Glutaminosaure Dehydrogenase aus Hefe. Biochem Z 328:361–367
    [Google Scholar]
  18. Hummelt G., Mora J. 1980; Regulation and function of glutamate synthase in Neurospora crassa. Biochem Biophys Res Commun 96:1688–1694 [CrossRef]
    [Google Scholar]
  19. Macheda L., Hynes M. J., Davis M. A. 1999; The Aspergillus nidulans gltA gene encoding glutamate synthase is required for ammonium assimilation in the absence of NADP-glutamate dehydrogenase. Curr Genet 34:467–471 [CrossRef]
    [Google Scholar]
  20. Maniatis T., Fritsch E. F., Sambrook J. 1982; Molecular Cloning. a Laboratory Manual. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  21. Marqués S., Florencio F. J., Candau P. 1992; Purification and characterization of the ferredoxin-glutamate synthase from the unicellular cyanobacterium Synechococcus sp. PCC 6301. Eur J Biochem 206:69–77 [CrossRef]
    [Google Scholar]
  22. Miflin B. J., Lea P. J., Wallsgrove R. M. 1980; The role of glutamine in ammonium assimilation and reassimilation in plants. In Glutamine: Metabolism, Enzymology and Regulation pp. 213–234Edited by Mora J., Palacios R. NY: Academic Press;
    [Google Scholar]
  23. Miller R. E., Stadtman E. R. 1972; Glutamate synthase from Escherichia coli. An iron–sulfide flavoprotein. J Biol Chem 247:7407–7419
    [Google Scholar]
  24. Nasmyth K. A., Reed S. I. 1980; Isolation of genes by complementation in yeast: molecular cloning of a cell-cycle gene. Proc Natl Acad Sci USA 77:2119–2123 [CrossRef]
    [Google Scholar]
  25. Ratti S., Curti B., Zanetti G., Galli E. 1985; Purification and characterization of glutamate synthase from Azospirillum brasilense. J Bacteriol 163:724–729
    [Google Scholar]
  26. Romero D., Dávila G. 1986; Genetic and biochemical identification of the glutamate synthase structural gene in Neurospora crassa. J Bacteriol 167:1043–1047
    [Google Scholar]
  27. Roon R. J., Even H. L., Larimore F. 1974; Glutamate synthase: properties of the reduced nicotinamide adenine dinucleotide-dependent enzyme from Saccharomyces cerevisiae. J Bacteriol 118:89–95
    [Google Scholar]
  28. Schägger H., Link T. A., Engel W. D., von Jagow G. 1986; Isolation of the eleven protein subunits of the bc1 complex from beef heart. Methods Enzymol 126:224–237
    [Google Scholar]
  29. Schreier H. J., Bernlohr R. W. 1984; Purification and properties of glutamate synthase from Bacillus licheniformis. J Bacteriol 160:591–599
    [Google Scholar]
  30. Senior P. J. 1975; Regulation of nitrogen metabolism in Escherichia coli and Klebsiella aerogenes: studies with the continuous culture technique. J Bacteriol 123:407–418
    [Google Scholar]
  31. Struhl K., Davis R. W. 1981; Transcription of the his3 gene region in Saccharomyces cerevisiae. J Mol Biol 152:535–552 [CrossRef]
    [Google Scholar]
  32. Tempest D. W., Meers J. L., Brown C. M. 1970; Synthesis of glutamate in Aerobacter aerogenes by a hitherto unknown route. Biochem J 117:405–407
    [Google Scholar]
  33. Towbin H., Staehelin T., Gordon J. 1979; Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76:4350–4354 [CrossRef]
    [Google Scholar]
  34. 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 glutamide amidotransferase. Proc Natl Acad Sci USA 71:4607–4611 [CrossRef]
    [Google Scholar]
  35. Tyler B. 1978; Regulation of the assimilation of nitrogen compounds. Annu Rev Biochem 47:1127–1162 [CrossRef]
    [Google Scholar]
  36. Valenzuela L., Guzmán-León S., Coria R., Ramı́rez J., Aranda C., González A. 1995; A NADP-glutamate dehydrogenase mutant of the petit-negative yeast Kluyveromyces lactis uses the glutamine synthethase-glutamate synthase pathway for glutamate biosynthesis. Microbiology 141:2443–2447 [CrossRef]
    [Google Scholar]
  37. Valenzuela L., Ballario P., Aranda C., Filetici P., González A. 1998; Regulation of the expression of GLT1, the gene encoding glutamate synthase in Saccharomyces cerevisiae. J Bacteriol 180:3533–3540
    [Google Scholar]
  38. Vanoni M. A., Negri A., Zanetti G., Ronchi S., Curti B. 1990; Structural studies on the subunits of glutamate synthase from Azospirillum brasilense. Biochim Biophys Acta 1039:374–377 [CrossRef]
    [Google Scholar]
  39. Wach A., Brachat A., Pohlmann R., Philippsen P. 1994; New heterologous modules for classical or PCR-based gene disruptions in Saccharomyces cerevisiae. Yeast 10:1793–1808 [CrossRef]
    [Google Scholar]
  40. Wolfe K. H., Shields D. C. 1997; Molecular evidence for an ancient duplication of the entire yeast genome. Nature 387:708–713 [CrossRef]
    [Google Scholar]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-146-1-239
Loading
/content/journal/micro/10.1099/00221287-146-1-239
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error