Identification by mutational analysis of four critical residues in the molybdenum cofactor domain of eukaryotic nitrate reductase.
The nucleotide sequence of the nitrate reductase (NR) molybdenum cofactor (MoCo) domain was determined in four Nicotiana plumbaginifolia mutants affected in the NR apoenzyme gene. In each case, missense mutations were found in the MoCo domain which affected amino acids that were conserved not only among eukaryotic NRs but also in animal sulfite oxidase sequences. Moreover an abnormal NR molecular mass was observed in three mutants, suggesting that the integrity of the MoCo domain is essential for a proper assembly of holo-NR. These data allowed to pinpoint critical residues in the NR MoCo domain necessary for the enzyme activity but also important for its quaternary structure.
Meyer
C
C
Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Versailles, France.
Gonneau
M
M
Caboche
M
M
Rouzé
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D002850:Chromatography, Gel; D003067:Coenzymes; D004252:DNA Mutational Analysis; D018076:DNA, Complementary / Q000737:chemistry; D008667:Metalloproteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D000090006:Molybdenum Cofactors; D009154:Mutation; D009243:NAD / Q000378:metabolism; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D010947:Plants, Toxic; D011487:Protein Conformation; D011621:Pteridines / Q000378:metabolism*; D014026:Nicotiana / Q000201:enzymology* / Q000235:genetics
D003067:Coenzymes; D018076:DNA, Complementary; D008667:Metalloproteins; D000090006:Molybdenum Cofactors; D009243:NAD; D050901:Nitrate Reductase; D009565:Nitrate Reductases; D011621:Pteridines; C022261:molybdopterin