Abstract
Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP+ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for α-ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP+-GDH activity (deamination). The results showed that NADPH-GDH and NADP+-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn+2 while the biosynthetic activity of the enzyme was dependent on Mg2+ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required α-ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP+ and NAD+, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.
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Abbreviations
- GDH:
-
glutamate dehydrogenase
- GOGAT:
-
glutamate synthase
- GS:
-
glutamine synthetase
References
Alvarez ME, McCarthy CM (1984) Glutamine synthetase from Mycobacterium avium. Can J Microbiol 30: 353–359
Blanco F, Alana A, Llama MJ, Serra JL (1989) Purification and properties of glutamine synthetase from the non-N2-fixing cyanobacterium Phormidium aminosum. J Bacteriol 171: 1158–1165
Caballero FJ, Igeno I, Cardenas J, Castillo F (1989) Regulation of reduced nitrogen assimilation in Rhodobacter capsulatus E1F1. Arch Microbiol 152: 508–511
Caughey WS, Smiley JD, Hellerman L (1954) l-Glutamic acid dehydrogenase: Structural requirements for substrate competition: effect of thyroxine. J Biol Chem 224: 591–607
Deuel TF, Turner DC (1972) Bacillus subtilis glutamine synthetase. Dependence of γ-glutamyltransferase activity on ionic strength and specific monovalent cations. J Biol Chem 247: 3039–3047
Farnden KJF, Robertson JG (1980) Methods for studying enzymes involved in metabolism related to nitrogenase. In: Bergersen FJ (ed) Methods for evaluating biological nitrogen fixation. John Wiley and Sons, Chichester New York Brisbane, pp 265–314
Florencio FJ, Ramos JL (1985) Purification and characterization of glutamine synthetase from the unicellular cyanobacterium Anacystis nidulans. Biochim Biophys Acta 838: 39–48
Hirose Y, Okada H (1979) Microbial production of amino acids. In: Pepler HJ, Perlman D (eds) Microbial technology, 2nd edn, vol 1. Academic Press, New York San Francisco London, pp 211–241
Hooper AB, Hansen J, Bell R (1967) Characterization of glutamate dehydrogenase from the ammonia-oxidizing chemoautotrop Nitrosomonans europaea. J Biol Chem. 242: 288–296
Janssen DB, Op den Camp HJM, Leenen PJM, Van derDrift Ch (1980) The enzymes of the ammonia assimilation in Pseudomonas aeruginosa. Arch Microbiol 124: 197–203
Khanna S, Nicholas DJD (1983) Some properties of glutamine synthetase and glutamate synthase from Chlorobium vibrioforme f. thiosulfatophilum. Arch Microbiol 134: 98–103
Kinoshita S (1985) Glutamic acid bacteria. In: Demain AL, Solomon NA (eds) Biology of industrial microorganisms. Benjamin/Cummings Publishing, London Amsterdam Don Mills, Ontario Sydney Tokyo, pp 115–143
Krishnan IS, Singhal RK, Dua RD (1986) Purification and characterization of glutamine synthetase from Clostridium pasteurianum. Biochemistry 25: 1589–1599
Kumar S, Nicholas DJD (1984) NAD+-and NADP+-dependent glutamate dehydrogenase in Nitrobacter agilis. J Gen Microbiol 130: 967–973
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193: 265–275
Matsuoka K, Kimura K (1985) Conformational changes in Mycobacterium smegmatis glutamine synthetase induced by certain divalent cations. J Biochem 97: 1033–1042
Matsuoka K, Kimura K (1986) Glutamate synthase from Bacillus subtilis PCI219. J Biochem 99: 1087–1100
Murrel JC, Dalton H (1983) Purification and properties of glutamine synthetase from Methylococcus capsulatus (Bath). J Gen Microbiol 129: 1187–1196
Phibbs JR, Bernlohr RW (1971) Purification, properties, and regulation of glutamic dehydrogenase of Bacillus licheniformis. J Bacteriol 106: 373–385
Schreier HJ, Bernlohr RW (1984) Purification and properties of glutamate synthase from Bacillus licheniformis. J Bacteriol 160: 591–599
Shiio I, Ozaki H (1970) Regulation of nicotinamide adenine dinucleotide phosphate-specific glutamate dehydrogenase from Brevibacterium flavum a glutamate-producing bacterium. J Bacteriol 68: 633–647
Singh P, Venkitasubramanian TA (1977) Glutamate dehydrogenase of Mycobacterium smegmatis. Ind J Biochem Biophys 14: 379–381
Singhal RK, Krishnan IS, Dua RD (1989) Stabilization, purification, and characterization of glutamate synthase from Clostridium pasteurianum. Biochemistry 28: 7928–7935
Stacey G, VanBaalen C, Tabita FR (1979) Nitrogen and ammonia assimilation in the cyanobacteria: regulation of glutamine synthetase. Arch Biochem Biophys 194: 457–467
Sung H-C, Tachiki T, Kumagai H, Tochikura T (1984) Production and preparation of glutamate synthase from Brevibacterium flavum. J Ferment Technol 62: 371–376
Tachiki T, Wakisaka S, Kumagai H, Tochikura T (1981) Glutamine synthetase from Micrococcus glutamicus: effect of nitrogen sources in culture medium on enzyme formation and some properties of crystalline enzyme. Agric Biol Chem 45: 1487–1492
Tachiki T, Sugh H-C, Wakisaka S, Tochikura T (1983a) Purification and some properties of glutamate synthase from Gluconobacter suboxydans grown on glutamate as a nitrogen source. J Ferment Technol 61: 179–184
Tachiki T, Wakisaka S, Suzuki H, Kumagai H, Tochikura T (1983b) Variation of properties of Micrococcus glutamicus glutamine synthetase brought about by divalent cations. Agric Biol Chem 47: 287–292
Tochikura T, Sung H-C, Tachiki T, Kumagai H (1984) Occurrence of glutamate synthase in Brevibacterium flavum. Agric Biol Chem 48: 2149–2150
Tyler B (1978) Regulation of the assimilation of nitrogen compounds. In: Snell EE, Boyer PD, Meister A, Richardson CC (eds) Annual review of biochemistry, vol 47. Annual Review Inc., California, pp 1147–1162
Vairinhos F, Bhandari B, Nicholas DJD (1983) Glutamine synthetase, glutamate synthase and glutamate dehydrogenase in Rhizobium japonicum strains grown in cultures and in bacteroids from root nodules of Glycine max. Planta 159: 207–215
Vandecasteele JP, Lemal J, Coudert M (1975) Pathways and regulation of glutamate synthesis in a Corynebacterium sp. overproducing glutamate. J Gen Microbiol 90: 178–180
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Ertan, H. Some properties of glutamate dehydrogenase, glutamine synthetase and glutamate synthase from Corynebacterium callunae . Arch. Microbiol. 158, 35–41 (1992). https://doi.org/10.1007/BF00249063
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DOI: https://doi.org/10.1007/BF00249063