HORT640 - Metabolic Plant Physiology
Ammonia Assimilation and Recycling
In certain fungi the main pathway of ammonia assimilation into glutamate involves the catalytic activity of NADPH-dependent glutamate dehydrogenase (GDH) [EC 184.108.40.206] (Sims and Folkes, 1964; Fincham, 1951):
NH3 + 2-oxoglutarate + NADPH + H+ <---> glutamate + NADP+
Direct ammonia assimilation into glutamate via the catalytic action of NADPH-GDH was demonstrated in the food yeast Candida utilis by 15NH3 tracer experiments and quantitative analysis of metabolic fluxes (Sims and Folkes, 1964).
In the filamentous fungus Neurospora crassa NADPH-GDH is encoded by the am gene (Kinsey et al, 1980). In Aspergillus nidulans the NADPH-GDH is designated gdhA (Kinghorn and Pateman, 1975; Gurr et al, 1986; Hawkins et al, 1989), while in Saccharomyces cerevisiae this gene is designated GDH1 (Moye et al, 1985). These NADPH-GDHs are hexamers with subunit molecular weights of ~ 50 kDa (Britton et al, 1992).
In fungi and yeasts a second NADH-dependent GDH [EC 220.127.116.11] functions primarily in glutamate catabolism (Sanwal and Lata, 1961):
glutamate + NAD+ <---> 2-oxoglutarate + NH3 + NADH + H+
The NADH-GDH of the filamentous fungus Neurospora crassa is encoded by the gene gdh (Kapoor et al, 1993). The equivalent enzyme in Aspergillus nidulans is encoded by gdhB (Kinghorn and Pateman, 1976). The NADH-GDHs of fungi and yeasts appear to be tetramers with much larger subunit size (~ 115 kDa) than the hexameric NADPH-GDHs involved in ammonia assimilation (Britton et al, 1992). The NADH-GDH of Candida utilis and Saccharomyces cerevisiae is regulated by reversible deactivation involving phosphorylation/dephosphorylation of the enzyme (Hemmings and Sims, 1977; Hemmings, 1978; 1981; Uno et al, 1984). The NADH-GDH of Neurospora crassa is regulated by catabolite repression (Kapoor et al, 1993).
Most bacteria (and cyanobacteria) possess a single NADPH-GDH isoform of the hexameric type: see e.g. Corynebacterium glutamicum [gdh] (Bormann et al, 1992); Escherichia coli [gdhA] (McPherson and Wootton, 1983); Salmonella typhimurium [gdhA] (Miller and Brenchley, 1984); and Synechocystis PCC 6803 [gdhA] (Chavez et al, 1995). Like the NADPH-GDHs of fungi and yeasts, these bacterial NADPH-GDHs are thought to be primarily involved in ammonia assimilation rather than glutamate catabolism. However, Bacteroides fragilis Bf1 has two GDH activities. One is dual cofactor NAD(P)H-dependent (encoded by gdhA), while the other has NADH-specific activity (encoded by gdhB) (Abrahams and Abratt, 1998). The dual coenzyme-specific GDH is regulated by reversible inactivation in vivo and repression by ammonium in Bacteroides fragilis (Yamamoto et al, 1987). In contrast to yeast, the mechanism of reversible inactivation of Bacteroides fragilis NAD(P)H does not appear to involve phosphorylation (Yamamoto et al, 1987). The dual specific NAD(P)H-GDH of Bacteroides thetaiotaomicron encoded by gdhA is typical of the family I-type hexameric GDH proteins (Baggio and Morrison, 1996). The Antarctic bacterium Psychrobacter sp. TAD1 also contains two distinct glutamate dehydrogenases (GDH), each specific for either NADPH or NADH (Di Fraia et al, 2000). The NADPH-GDH of Psychrobacter has a hexameric structure (Di Fraia et al, 2000).
Two distinct genes (designated GDH1 and GDH2) encoding NADH-GDH have been identified in Arabidopsis thaliana (Turano et al, 1997). Both gene products contain putative mitochondrial transit polypeptides and NADH- and 2-oxoglutarate-binding domains (Turano et al, 1997). Subcellular fractionation confirms the mitochondrial location of the NADH-GDH isoenzymes (Turano et al, 1997). GDH1 encodes a 43.0 kDa polypeptide, designated alpha, and GDH2 encodes a 42.5 kDa polypeptide, designated beta (Turano et al, 1997). The two subunits combine in different ratios to form seven NADH-GDH isoenzymes (Turano et al, 1997). In Arabidopsis, the slowest-migrating isoenzyme in a native gel, GDH1, is a homohexamer composed of alpha subunits, and the fastest-migrating isoenzyme, GDH7, is a homohexamer composed of beta subunits (Turano et al, 1997). GDH isoenzymes 2 through 6 are heterohexamers composed of different ratios of alpha and beta subunits (Turano et al, 1997).
Zea mays also possesses two distinct loci encoding NADH-GDH (Pryor, 1990). Consistent with the Arabidopsis model described above (Turano et al, 1997), maize mutants deficient in gdh1 (Pryor, 1990) show only a single GDH isozyme corresponding to isozyme 7 [i.e. the homohexamer derived from the gdh2 gene product] (Magalhaes et al, 1990). The wildtype shows seven isozymes:
Native gel of GDH isozymes of the maize gdh1-null mutant (left) and wildtype (right). Root extracts were subjected to gel electrophoresis and stained for GDH as described by Magalhaes et al (1990).
(see also discussion of GDH under gamma-aminobutyrate (GABA) metabolism in the Aminotransferase reactions section)
In contrast to higher plants Chlorella sorokiniana has seven ammonium-inducible, chloroplastic NADPH-GDH isozymes composed of varying ratios of alpha- and beta-subunits (Miller et al, 1998). The C. sorokiniana genome possesses a single 7178 bp nuclear NADPH-GDH gene (Miller et al, 1998). This single gene produces two 2074 and 2116 nucleotide mRNAs encoding precursor proteins of 56,350 and 57,850 Da, respectively (Miller et al, 1998). The two NADPH-GDH mRNAs are identical with the exception of a 42 nucleotide sequence located within the 5'-coding region of the longer mRNA (Miller et al, 1998). This 42 nucleotide appears to undergo alternative splicing from the precursor mRNA by a process that is regulated by both nutritional and environmental signals (Miller et al, 1998). The mature alpha- and beta-subunits of 53,501 and 52,342 Da are identical in sequence except for an 11 amino acid extension at the N-terminus of the alpha-subunit (Miller et al, 1998).
Abrahams GL, Abratt VR 1998 The NADH-dependent glutamate dehydrogenase enzyme of Bacteroides fragilis Bf1 is induced by peptides in the growth medium. Microbiology 144: 1659-1667.
Baggio L, Morrison M 1996 The NAD(P)H-utilizing glutamate dehydrogenase of Bacteroides thetaiotaomicron belongs to enzyme family I, and its activity is affected by trans-acting gene(s) positioned downstream of gdhA. J. Bacteriol. 178: 7212-7220.
Britton KL, Baker PJ, Rice DW, Stillman TJ 1992 Structural relationship between the hexameric and tetrameric family of glutamate dehydrogenases. Eur. J. Biochem. 209: 851-859.
Chavez S, Reyes JC, Chauvat F, Florencio FJ, Candau P 1995 The NADP-glutamate dehydrogenase of the cyanobacterium Synechocystis 6803: cloning, transcriptional analysis and disruption of the gdhA gene. Plant Mol. Biol. 28: 173-188.
Di Fraia R, Wilquet V, Ciardiello MA, Carratore V, Antignani A, Camardella L, Glansdorff N, Di Prisco G 2000 NADP+-dependent glutamate dehydrogenase in the Antarctic psychrotolerant bacterium Psychrobacter sp. TAD1. Characterization, protein and DNA sequence, and relationship to other glutamate dehydrogenases. Eur. J. Biochem. 267: 121-131.
Fincham JRS 1951 The occurrence of glutamate dehydrogenase in Neurospora and its apparent absence in certain mutant strains. J. Gen. Microbiol. 5: 793-806.
Gurr SJ, Hawkins AR, Drainas C, Kinghorn JR 1986 Isolation and identification of the Aspergillus nidulans gdhA gene encoding NADP-linked glutamate dehydrogenase. Curr. Genet. 10: 761-766.
Hawkins AR, Gurr SJ, Montague P, Kinghorn JR 1989 Nucleotide sequence and regulation of expression of the Aspergillus nidulans gdhA gene encoding NADP dependent glutamate dehydrogenase. Mol. Gen. Genet. 218: 105-111.
Hemmings BA 1978 Phosphorylation of NAD-dependent glutamate dehydrogenase from yeast. J. Biol. Chem. 253: 5255-5258.
Hemmings BA 1981 Reactivation of the phospho form of the NAD-dependent glutamate dehydrogenase by a yeast protein phosphatase. Eur. J. Biochem. 116: 47-50.
Hemmings BA, Sims AP 1977 The regulation of glutamate metabolism in Candida utilis. Evidence for two interconvertible forms of NAD-dependent glutamate dehydrogenase. Eur. J. Biochem. 80: 143-151.
Kapoor M, Vijayaraghavan Y, Kadonaga R, LaRue KE 1993 NAD(+)-specific glutamate dehydrogenase of Neurospora crassa: cloning, complete nucleotide sequence, and gene mapping. Biochem. Cell Biol. 71: 205-219.
Kinghorn JR, Pateman JA 1975 The structural gene for NADP L-glutamate dehydrogenase in Aspergillus nidulans. J. Gen. Microbiol. 86: 294-300.
Kinsey JA, Fincham JR, Siddig MA, Keighren M 1980 New mutational variants of Neurospora NADP-specific glutamate dehydrogenase. Genetics 95: 305-316.
Magalhaes JR, Ju GC, Rich PJ, Rhodes D 1990 Kinetics of 15NH4+ assimilation in Zea mays: Preliminary studies with a glutamate dehydrogenase (GDH1) null mutant. Plant Physiol. 94: 647-656.
McPherson MJ, Wootton JC 1983 Complete nucleotide sequence of the Escherichia coli gdhA gene. Nucleic Acids Res. 11: 5257-5266.
Miller ES, Brenchley JE 1984 Cloning and characterization of gdhA, the structural gene for glutamate dehydrogenase of Salmonella typhimurium. J. Bacteriol. 157: 171-178.
Miller PW, Dunn WI, Schmidt RR 1998 Alternative splicing of a precursor-mRNA encoded by the Chlorella sorokiniana NADP-specific glutamate dehydrogenase gene yields mRNAs for precursor proteins of isozyme subunits with different ammonium affinities. Plant Mol. Biol. 37: 243-63.
Moye WS, Amuro N, Rao JK, Zalkin H 1985 Nucleotide sequence of yeast GDH1 encoding nicotinamide adenine dinucleotide phosphate-dependent glutamate dehydrogenase. J. Biol. Chem. 260: 8502-8508.
Pryor A 1990 A maize glutamate dehydrogenase null mutant is cold temperature sensitive. Maydica 35: 367-372.
Sanwal BD, Lata M 1961 Glutamate dehydrogenase in single gene mutants of Neurospora deficient in amination. Nature (Lond.) 190: 286-287.
Sims AP, Folkes BF 1964 A kinetic study of the assimilation of 15N-ammonia and the synthesis of amino acids in an exponentially growing culture of Candida utilis. Proc. Roy. Soc. Lond. B. Biol. Sci. 159: 479-502.
Turano FJ, Thakkar SS, Fang T, Weisemann JM 1997 Characterization and expression of NAD(H)-dependent glutamate dehydrogenase genes in Arabidopsis. Plant Physiol. 113: 1329-1341.
Uno I, Matsumoto K, Adachi K, Ishikawa T 1984 Regulation of NAD-dependent glutamate dehydrogenase by protein kinases in Saccharomyces cerevisiae. J. Biol. Chem. 259: 1288-1293.
Yamamoto I, Saito H, Ishimoto M 1987 Regulation of synthesis and reversible inactivation in vivo of dual coenzyme-specific glutamate dehydrogenase in Bacteroides fragilis. J. Gen. Microbiol. 133: 2773-2780.
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