NAD+-dependent glutamate dehydrogenase of the edible mushroom Agaricus bisporus: biochemical and molecular characterization

Abstract

The NAD+-dependent glutamate dehydrogenase (NAD-GDH) of Agaricus bisporus, a key enzyme in nitrogen metabolism, was purified to homogeneity. The apparent molecular mass of the native enzyme is 474 kDa comprising four subunits of 116 kDa. The isoelectric point of the enzyme is about 7.0. Km values for ammonium, 2-oxoglutarate, NADH, glutamate and NAD+ were 6.5, 3.5, 0.06, 37.1 and 0.046 mM, respectively. The enzyme is specific for NAD(H). The gene encoding this enzyme (gdhB) was isolated from an A. bisporus H39 recombinant lambda phage library. The deduced amino acid sequence specifies a 1029-amino acid protein with a deduced molecular mass of 115,463 Da, which displays a significant degree of similarity with NAD-GDH of Saccharomyces cerevisiae and Neurospora crassa. The ORF is interrupted by fifteen introns. Northern analysis combined with enzyme activity measurements suggest that NAD-GDH from A. bisporus is regulated by the nitrogen source. NAD-GDH levels in mycelium grown on glutamate were higher than NAD-GDH levels in mycelium grown on ammonium as a nitrogen source. Combined with the kinetic parameters, these results suggest a catabolic role for NAD-GDH. However, upon addition of ammonium to the culture transcription of the gene is not repressed as strongly as that of the gene encoding NADP-GDH (gdhA). To date, tetrameric NAD-GDHs with large subunits, and their corresponding genes, have only been isolated from a few species. This enzyme represents the first NAD-GDH of basidiomycete origin to be purified and is the first such enzyme from basidiomycetes whose sequence has been determined.

Expression of the urease gene of Agaricus bisporus: a tool for studying fruit body formation and post-harvest development

“…The decrease in urea content of the growing sporophore was thus not only the result of the dilution effect as the fruit body develops and grows to a larger volume. The urease activity demonstrated indicates that ammonium is produced and can be reassimilated by the primary ammonium assimilation pathways addressed by Baars et al (1996) and Kersten et al (1997Kersten et al ( , 1998Kersten et al ( , 1999. For mature fruit bodies of A. bisporus, however, no reassimilation of urea-ammonia was determined as was shown for puffballs of Lycoperdon pyriforme (Reinbothe et al 1967).…”

“…The PCR product of forward primer 5′-GGC GAC GAA GTC AAA TTT GG-3′ (ure1f) and reverse primer 5′-TCG TTG AGA GTG TCG GTATG-3′ (ure2r) was labelled with [ 32 P] CTP and used as a probe (Wagemaker et al 2005). Northern blots were also probed with an A. bisporus 28S ribosomal DNA fragment as loading control and with probes specific for the genes gdhA, gdhB and glnA encoding enzymes of the primary nitrogen metabolism (Kersten et al 1997(Kersten et al , 1999.…”

“…The slight increase at 15-18 h is not significant when the signals are corrected for the 28S rRNA loading control. For comparison, the blot was also hybridized with probes specific for the NADP-dependent GDHs (gdhA, gdhB) and the glutamine synthetase (glnA) of A. bisporus (Kersten et al 1997(Kersten et al , 1999. These enzymes of the primary nitrogen metabolism showed the same expression pattern (Fig.…”

Biochemical and molecular characterization of NADP‐glutamate dehydrogenase from the ectomycorrhizal fungus Tuber borchii

“…This evidence suggested similar conformations for these enzymes (Blumenthal et al ., 1975) and a common evolutionary origin. Other GDHs such as NAD‐dependent GDH purified from Saccaromyces cerevisae (Uno et al ., 1984), N. crassa (Veronese et al ., 1974), Agaricus bisporus (Kersten et al ., 1999) and L. bicolor (Garnier et al ., 1997) have a tetrameric structure with subunits of 115–116 kDa.…”

Structural and functional properties of glutamate dehydrogenases.

“…Abbreviations: ELFV, a domain common to glu/leu/phe/val dehydrogenases (the substrate-binding domain); NAD, a NAD-binding Rossmann-like domain (use of 'NAD' intends no implication on cofactor specificity). Agaricus bisporus (Kersten et al, 1999) NAD + 3.5 37.1 6.5 46 60…”