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

The NAD(+)-dependent glutamate dehydrogenase (NAD-GDH) of Agaricus bisporus , a key enzyme in nitrogen metabolism, was purified to homogeneity. The app arent molecular mass of the native enzyme is 474 kDa comprising four subuni ts of 116 kDa. The isoelectric point of the enzyme is about 7.0. K-m 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 reco mbinant lambda phage library. The deduced amino acid sequence specifies a 1 029-amino acid protein with a deduced molecular mass of 115,463 Da, which d isplays a significant degree of similarity with NAD-GDH of Saccharomyces ce revisiae and Neurospora crassa. The ORF is interrupted by fifteen introns. Northern analysis combined with enzyme activity measurements suggest that N AD-GDH from A. bisporus is regulated by the nitrogen source. NAD-GDH levels in mycelium grown on glutamate were higher than NADGDH levels in mycelium grown on ammonium as a nitrogen source. Combined with the kinetic parameter s, these results suggest a catabolic role for NAD-GDH. However, upon additi on 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 bee n 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 basid iomycetes whose sequence has been determined.