Functional Dissection and Site-directed Mutagenesis of the Structural Gene for NAD(P)H-Nitrite Reductase in Neurospora crassa
| Autor: | Reginald H. Garrett, James D. Colandene |
|---|---|
| Rok vydání: | 1996 |
| Předmět: |
7-Dehydrocholesterol reductase
Antigens Fungal DNA Complementary Nitrite Reductases Blotting Western Genes Fungal Molecular Sequence Data Heme Biology Reductase Biochemistry Neurospora crassa Fungal Proteins Structure-Activity Relationship chemistry.chemical_compound Siroheme Amino Acid Sequence RNA Messenger Site-directed mutagenesis Molecular Biology Nitrite Reductase (NAD(P)H) Binding Sites Structural gene RNA Fungal Cell Biology NAD Nitrite reductase biology.organism_classification Molecular biology Recombinant Proteins chemistry Flavin-Adenine Dinucleotide Mutagenesis Site-Directed NAD+ kinase |
| Zdroj: | Journal of Biological Chemistry. 271:24096-24104 |
| ISSN: | 0021-9258 |
| DOI: | 10.1074/jbc.271.39.24096 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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