Abstract
Nitrate reductase is a multiredox enzyme possessing three functional domains associated with the prosthetic groups FAD, heme iron, and molybdopterin. In Aspergillus nidulans, it is encoded by the niaD gene. A homologous transformation system has been used whereby a major deletion at the niiAniaD locus of the host was repaired by gene replacement. Employing site-directed mutagenesis and this transformation system, nine niaD mutants were generated carrying specific amino acid substitutions. Mutants in which alanine replaced cysteine 150, which is thought to bind the molybdenum atom of the molybdenum-pterin, and in which alanine replaced histidine 547, which putatively binds heme iron, had no detectable nitrate reductase (NAR) activity. This clearly establishes an essential catalytic role for these residues. Of the remaining mutants, all altered in the NADPH/FAD domain, two were temperature-sensitive for NAR activity, two had reduced NAR activity levels, and three had normal levels. Since some of these mutants change residues conserved between homologous nitrate reductases from a wide range of species, it is clear that such amino acid identities do not necessarily signify essential roles for the activity of the enzyme. These findings are considered in the light of predicted structural/functional roles for the altered amino acids.
Highlights
Nitrate reductase is a multiredox enzyme possessing three functional domains associated with the prosthetic groups FAD, heme iron, and molybdopterin
Since some of these mutants change residues conserved between homologous nitrate reductases from a wide range of species, it is clear that such amino acid identities do not necessarily signify essential roles for the activity of the enzyme
NiaDC150A occurs at a cysteine residue within the MoCo domain predicted to form a ligand with the molybdenum atom [19]. niaDH547A occurs at the putative first axial histidine of the heme domain presumed to bind to heme iron [20,21,22]. niaDW618K changes a residue conserved in fungi to one conserved in plants. niaDK626A is at a site conserved in all nitrate reductase (NAR) molecules [9]. niaDH654A alters a residue implicated in NAD(PlH binding [24, 25]. niaDY693A alters a residue implicated in FAD binding [23]. niaDG721S was made to mimic a naturally isolated mutation so that the identity of the transgenic and natural mutations could be assessed
Summary
Nitrate reductase is a multiredox enzyme possessing three functional domains associated with the prosthetic groups FAD, heme iron, and molybdopterin. All altered in the NADPHIFAD domain, two were temperature-sensitive for NAR activity, two had reduced NAR activity levels, and three had normal levels Since some of these mutants change residues conserved between homologous nitrate reductases from a wide range of species, it is clear that such amino acid identities do not necessarily signify essential roles for the activity of the enzyme. Inorganic nitrate can be used as a nitrogen source by fungi, algae, some yeasts, and some bacteria as well as by higher plants (see Ref. 1 and included reviews) These organisms perform nitrate assimilation as a sequential reduction of NOi to N02' and N02'to NUt by the action of two enzymes nitrate reductase (NAR)l and nitrite reductase. The partial activities include the reduction of NOi by electrons passed to it from reduced methyl viologen (MVH) through Heme and MoCo, or bromphenol blue (BPB) through only MoCo [6, 7]
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