Regulation of Glutamate Dehydrogenases in nit-2 and am Mutants of Neurospora crassa

Abstract

The regulation of the glutamate dehydrogenases was investigated in wild-type Neurospora crassa and two classes of mutants altered in the assimilation of inorganic nitrogen, as either nitrate or ammonium. In the wild-type strain, a high nutrient carbon concentration increased the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-glutamate dehydrogenase and decreased the activity of reduced nicotinamide adenine dinucleotide (NADH)glutamate dehydrogenase. A high nutrient nitrogen concentration had the opposite effect, increasing NADH-glutamate dehydrogenase and decreasing NADPHglutamate dehydrogenase. The nit-2 mutants, defective in many nitrogen-utilizing enzymes and transport systems, exhibited low enzyme activities after growth on a high sucrose concentration: NADPH-glutamate dehydrogenase activity was reduced 4-fold on NH4Cl medium, and NADH-glutamate dehydrogenase, 20-fold on urea medium. Unlike the other affected enzymes of nit-2, which are present only in basal levels, the NADH-glutamate dehydrogenase activity was found to be moderately enhanced when cells were grown on a low carbon concentration. This finding suggests that the control of this enzyme in nit-2 is hypersensitive to catabolite repression. The am mutants, which lack NADPH-glutamate dehydrogenase activity, possessed basal levels of NADH-glutamate dehydrogenase activity after growth on urea or L-aspartic acid media, like the wild-type strain, and possessed moderate levels (although threeto fourfold lower than the wild-type strain) on L-asparagine medium or L-aspartic acid medium containing NH4C1. These regulatory patterns are identical to those of the nit-2 mutants. Thus, the two classes of mutants exhibit a common defect in NADH-glutamate dehydrogenase regulation. Double mutants of nit-2 and am had lower NADH-glutamate dehydrogenase activities than either parent. A carbon metabolite is proposed to be the repressor of NADH-glutamate dehydrogenase in N. crassa.

Regulation of nitrogen metabolism and gene expression in fungi.

“…This result clearly shows that the NAD-GDH enzyme can be turned on by carbon limitation, independent of any positive activation from the nitrogen circuit. The am mutant also shows decreased levels of NAD-GDH, similar to that observed with nit-2, however, in this case the effect could be secondary due to the accumulation in am of a carbon catabolite (e.g., a-ketoglutarate) which represses NAD-GDH synthesis (25).…”

“…Another complication which makes their study difficult is that these enzymes appear to be controlled by both the nitrogen and carbon circuits. High activity of NADP-GDH is found in Neurospora (25) and Aspergillus (51) when wild-type cells are grown with a limited amount of inorganic nitrogen source, such as nitrate or ammonia. Increasing amounts of inorganic nitrogen compounds repress NADP-GDH but cause an increase in NAD-GDH.…”

“…Thus, even in the presence of a rich carbon source (2% sucrose), NAD-GDH is increased approximately 10-fold during growth of wild-type, nit-i, or nit-3 Neurospora strains on 150 mM urea compared with growth on 25 mM NH4Cl; by contrast, three different nit-2 mutants possessed only the basal level of NAD-GDH under either condition. It therefore appears that NAD-GDH can also be derepressed by the nitrogen source under certain conditions and that the nit-2 control mutant is defective in this nitrogen response, but can derepress NAD-GDH via the carbon signal (25). Considerable work is needed to understand the mechanisms, which appear to be complex, that control NADP-GDH and NAD-GDH; one particularly interesting question which needs careful study is to determine whether there is any absolute connection which links the synthesis of these two enzymes, such that they are necessarily formed in a reciprocal fashion.…”

Role of the Regulatory Gene areA of Aspergillus oryzae in Nitrogen Metabolism

“…It is interesting that nit-2 mutants of N. crassa grow as well as the wild type on solid medium but produce less mycelial mass on ammonium in liquid medium (12,28). Furthermore, Dantzig et al (11) and Dunn-Coleman et al (12) found that nit-2 mutants produce only basal levels of NADP-GDH. Therefore, effects on ammonium utilization and NADP-GDH levels may be common features of the loss of the major nitrogen regulatory protein in other fungal species.…”

Nitrogen Source Regulates Glutamate Dehydrogenase NADP Synthesis in Neurospora crassa

“…Pateman has studied the changes in activity of this enzyme in A. nidulans and claims that N. crassa exhibits a similar pattern of regulation (17). Although there have been some reports on the effect of nitrogen nutrition on the activity of N. crassa GDH-NADP (5,9,24), there is not much information about the regulation of this enzyme (18). On the other hand, the detailed genetic and structural studies of Fincham and colleagues (3,10,12,25,26) have established the oligomeric structure and the amino acid sequence of the enzyme and have demonstrated the colinearity between the mutational sites and the amino acid positions in the polypeptide.…”