Oxidation of Neurospora crassa NADP-specific glutamate dehydrogenase by activated oxygen species

Abstract

The glutamine synthetase and the NADP-specific glutamate dehydrogenase activities of Neurospora crassa were lost in a culture without carbon source only when in the presence of air. Glutamine synthetase was previously reported to be liable to in vitro and in vivo inactivation by activated oxygen species. Here we report that NADP-specific glutamate dehydrogenase was remarkably stable in the presence of activated oxygen species but was rendered susceptible to oxidative inactivation when chelated iron was bound to the enzyme and either ascorbate or H202 reacted on the bound iron. This reaction gave rise to further modifications of the enzyme monomers by activated oxygen species, to partial dissociation of the oligomeric structure, and to precipitation and fragmentation of the enzyme. The in vitro oxidation reaction was affected by pH, temperature, and binding to the enzyme of NADPH. Heterogeneity in total charge was observed in the purified and immunoprecipitated enzymes, and the relative amounts of enzyme monomers with different isoelectric points changed with time of the oxidizing reaction.Modification of proteins by activated oxygen species has been identified in various enzymes (5, 13, 14, 17, 20, 26-28, 30, 41, 43, 46, 48, 51). This modification is generally accompanied by changes in catalytic activity, usually inhibiting it (5, 13, 14, 17, 20, 26-28, 41, 43), but there are also examples of enzyme activation mediated by highly reactive oxygen metabolites (30,48,51). Several oxidation systems have been used, including neutrophil oxidative burst (34, 39), enzymatic systems such as cytochrome P-450 (17, 44), and nonenzymatic systems such as the ascorbate model system (27). Modification of proteins by mixed-function oxidation has been found in pathological-related processes associated with oxygen toxocity (45), host defense mechanisms (34), and aging (35). The physiological functions of oxidation by activated oxygen species have not been defined yet, but they may include intracellular proteolysis (8,11,(37)(38)(39)(40) and regulation of anaerobic and aerobic oxidation metabolism (21). We have recently suggested that oxidation of proteins by activated oxygen species is also related to developmental processes in microorganisms (W. Hansberg and J. Aguirre, J. Theor. Biol., in press).Part of our research has focused on the regulation of nitrogen metabolism during germination and conidiation of Neurospora crassa (6,7,19). In the mycelium which forms the aerial hyphae (47), glutamine synthetase (GS) and NADP-specific glutamate dehydrogenase [GDH(NADP)] activities are lost (7). The N. crassa GS is modified in vitro by activated oxygen species (1) in a similar way to that of Escherichia coli GS, in which mixed-function oxidation has been studied in great detail (15,17,(26)(27)(28)32 extract is incubated with reduced NAD or NADP and (ii) when purified GS is incubated with hydrogen peroxide and ferrous iron (Fe2") or with ascorbate and ferric iron (Fe3") in the presence of oxygen (1). The in vitro-oxidized GS is completely...

Biochemistry and pathology of radical-mediated protein oxidation

“…Reversible elevation of protein carbonyl levels (from high basal values of around 10 nmol\mg of protein) by up to 2.5-fold in less than 1 h was observed in association with each transition [174]. Reversal also took less than 1 h. The cell-free oxidative inactivation of glutamate dehydrogenase and glutamine synthetase from the fungus [51,207] created more acidic polypeptides, and importantly such acidic species were also observed in i o and found to display enhanced susceptibility to proteinases. Loss of glutamine synthetase activity occurred shortly before each differentiation step, coincident with the increase in protein carbonyls [176].…”

Temporal and Spatial Regulation of Gene Expression During Asexual Development ofNeurospora crassa

“…The current hypothesis is that exposure of the mycelium to air induces a hyperoxidant state, which leads to the activation of cAMP and MAP kinase signal transduction pathways that trigger formation of aerial hyphae, which grow perpendicular to the substrate mycelium (Aguirre et al 1989(Aguirre et al , 2006Michan et al 2003). Aerial hyphae are thought to be physiologically different from the vegetative mycelium, and aerial hyphae-specific genes have been identified, which are not expressed in the substrate mycelium or in conidiophores (Li et al 2005).…”

Enzyme inactivation related to a hyperoxidant state during conidiation of Neurospora crassa

“…Enzyme determinations. Cell extracts for enzyme determination were prepared as described before Aguirre et a/., 1989); G S activity was determined by the method of Ferguson & Sims (1971). Transferase activity is expressed as pmol y-glutamyl hydroxamate produced at 30 "C (mg protein)-' min-l. G D H activities were measured following the oxidation of either NADPH for the biosynthetic enzyme or NADH for the catabolic one following the method of Fincham (1957).…”

“…However a site-specific oxidative modification occurred when a dicarboxylic acid chelating iron was bound to the allosteric site (Aguirre et al, 1989). Thus, oxidation of GDH(NADP) required an intracellular availability of free iron and dicarboxylic acids.…”

“…The oxidized G S was also shown to be more susceptible to attack by endogenous proteases. Oxidized polypeptides were detected in vivo under conditions in which these enzymes are degraded, and degradation of both was dependent on the presence of air Aguirre et a/., 1989). It was hypothesized that, in hyphae exposed directly to air, a hyperoxidant state developed, characterized by increased generation of reactive oxygen species in amounts surpassing the cellular competence to neutralize them.…”