High-level expression in Escherichia coli of the catalytically active flavin domain of corn leaf NADH:nitrate reductase and its comparison to human NADH:cytochrome B5 reductase.
Higher plant nitrate reductase can be divided into three functional domains representing its prosthetic groups: 1) flavin; 2) cytochrome b; and 3) Mo-pterin. The flavin domain has been synthesized by heterologous expression in Escherichia coli using a fragment of a corn leaf NADH:nitrate reductase cDNA clone, Zmnr1, which we had previously isolated and sequenced. A Xho2-BamH1 fragment was cut from Zmnr1, containing the sequence for the flavin domain, and ligated in the BamH1 site of expression vector pET3c. When this construct was expressed in E. coli, a 30 kD polypeptide was found to be newly synthesized. The flavin domain was purified to homogeneity using blue Sepharose and shown to have a molecular weight of 30 kD. The recombinant flavin domain has a ferricyanide reductase specific activity of 1000 mumols NADH oxidized/min/mg protein and a visible spectrum virtually identical to that of human NADH:cytochrome b5 reductase.
Hyde
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GE
Plant Biotechnology Group, Michigan Technological University, Houghton 49931.
Campbell
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WH
D000595:Amino Acid Sequence; D002384:Catalysis; D003579:Cytochrome Reductases / Q000235:genetics*; D042966:Cytochrome-B(5) Reductase; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000235:genetics*; D005415:Flavins / Q000235:genetics / Q000302:isolation & purification; D015870:Gene Expression; D006801:Humans; D008969:Molecular Sequence Data; D050895:Nitrate Reductase (NADH); D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism; D010452:Peptide Biosynthesis; D010455:Peptides / Q000302:isolation & purification; D011994:Recombinant Proteins; D012689:Sequence Homology, Nucleic Acid; D003313:Zea mays / Q000201:enzymology* / Q000235:genetics
D005415:Flavins; D010455:Peptides; D011994:Recombinant Proteins; D003579:Cytochrome Reductases; D042966:Cytochrome-B(5) Reductase; D009565:Nitrate Reductases; D050895:Nitrate Reductase (NADH)