title: Functional dissection and site-directed mutagenesis of the structural gene for NAD(P)H-nitrite reductase in Neurospora crassa.
abstract: Neurospora crassa NAD(P)H-nitrite reductase, encoded by the nit-6 gene, is a soluble, alpha2-type homodimeric protein composed of 127-kDa polypeptide subunits. This multicenter oxidation-reduction enzyme utilizes either NADH or NADPH as electron donor and possesses as prosthetic groups two iron-sulfur (Fe4S4) clusters, two siroheme groups, and two FAD molecules. The native activity of the enzyme is the NAD(P)H-dependent reduction of nitrite to ammonia. In addition, N. crassa nitrite reductase displays several partial activities in vitro, including a siroheme-independent NAD(P)H-cytochrome c reductase activity and an FAD-independent dithionite-nitrite reductase activity. These partial activities are presumed to be manifestations of discrete functional domains within the protein. A full-length nit-6 cDNA was constructed and used in developing an expression system within E. coli capable of yielding high levels of NADPH-nitrite reductase activity. Maximal expression was obtained in nirB- E. coli cells grown anaerobically at 22 +/- 1 degrees C, in conjunction with co-expression of a plasmid-borne cysG gene (encoding the rate-limiting enzyme in siroheme synthesis) and co-transformation with plasmid pGroESL (encoding bacterial chaperonins GroES and GroEL). Dissection of gene segments encoding putative functional domains within the nit-6 gene was performed. Expression of a partial cDNA construct encoding the FAD-/NAD-binding domain yielded extracts with NADPH-cytochrome c reductase activity but no NADPH-nitrite reductase activity or dithionite-nitrite reductase activity. Expression of a cDNA construct encoding the (Fe4S4)-siroheme-binding domain resulted in extracts possessing dithionite-nitrite reductase activity but no NADPH-nitrite reductase or NADPH-cytochrome c reductase activity. Analysis of site-directed mutations altering amino acid residues Cys-331 within the FAD-/NAD-binding domain and Ser-755 within the (Fe4S4)-siroheme-binding domain of the nitrite reductase demonstrated that these residues were not essential for native or partial enzyme activity. Cys-757 within the (Fe4S4)-siroheme-binding domain was essential for native enzyme activity.
authors.affiliation: Department of Biology, Gilmer Hall, University of Virginia, Charlottesville, Virginia 22903-2477, USA.
authors.forename: J D
authors.initials: JD
authors.lastname: Colandene
authors.forename: R H
authors.initials: RH
authors.lastname: Garrett
mesh_terms: D000595:Amino Acid Sequence; D000946:Antigens, Fungal / Q000737:chemistry; D001665:Binding Sites; D015153:Blotting, Western; D018076:DNA, Complementary / Q000235:genetics; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D005800:Genes, Fungal; D006418:Heme / Q000031:analogs & derivatives / Q000378:metabolism; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009243:NAD / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology* / Q000235:genetics; D050898:Nitrite Reductase (NAD(P)H); D009572:Nitrite Reductases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D012331:RNA, Fungal / Q000235:genetics; D012333:RNA, Messenger / Q000235:genetics; D011994:Recombinant Proteins; D013329:Structure-Activity Relationship
chemical_list: D000946:Antigens, Fungal; D018076:DNA, Complementary; D005656:Fungal Proteins; D012331:RNA, Fungal; D012333:RNA, Messenger; D011994:Recombinant Proteins; D009243:NAD; D005182:Flavin-Adenine Dinucleotide; D006418:Heme; C009272:siroheme; D009572:Nitrite Reductases; D050898:Nitrite Reductase (NAD(P)H)