Purification and characterization of the assimilatory NADPH-nitrate reductase of Aspergillus nidulans.
NADPH-nitrate reductase [NADPH : nitrate oxidoreductase, EC 1.6.6.3] was purified 500-fold from Aspergillus nidulans with an overall yield of about 20%. The purified enzyme catalyzed NADPH-nitrate, NADPH-cytochrome c, FADH2-nitrate and reduced methyl viologen-nitrate reductase activities. Its molecular weight was estimated to be 180,000 from the Stokes radius and sedimentation coefficient. The oxidized enzyme exhibited an absorption spectrum having a peak at 412 nm and a broad shoulder at about 450 nm. When reduced with NADPH, absorption peaks appeared at 423 (Soret), 527 (beta) and 557 (alpha) nm, and absorption in the 450 nm region decreased. Upon treatment of the reduced enzyme with KNO3, the spectrum returned to that of the oxidized enzyme. The presence of protoheme in the enzyme was confirmed by the absorption spectrum of reduced pyridine hemochromogen. It was concluded that a b-type cytochrome ("cytochrome b-557") is present in the enzyme and is involved in the intramolecular electron transport from NADPH to nitrate. The NADPH-nitrate and NADPH-cytochrome c reductase activities, but not the other two activities, were significantly decreased by incubation of the enzyme at 37 degrees C in the absence of FAD. Analysis by SDS slab gel electrophoresis suggested that the nitrate reductase consists of two each of two subunits of 59,000 and 38,000 daltons and that a dissociation of 38,000 subunits from the native enzyme occurs during heat treatment, resulting in alteration of the catalytic activity.
D001233:Aspergillus nidulans / Q000201:enzymology*; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis; D007700:Kinetics; D046911:Macromolecular Substances; D008970:Molecular Weight; D050896:Nitrate Reductase (NAD(P)H); D009565:Nitrate Reductases / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D013053:Spectrophotometry
D046911:Macromolecular Substances; D005182:Flavin-Adenine Dinucleotide; D009565:Nitrate Reductases; D050896:Nitrate Reductase (NAD(P)H)