Mutational and structural analysis of the nitrate reductase heme domain of Nicotiana plumbaginifolia.
We have analyzed four Nicotiana plumbaginifolia null mutants presumably affected in the heme domain of nitrate reductase. The DNA sequence of this domain has been determined for each mutant and for the wild type. Two mutations were identified as single base changes leading to, respectively, the substitution of a histidine residue by an asparagine (mutant E56) and to the appearance of an ochre stop codon (mutant E64). Based on the amino acid sequence homology between the nitrate reductase heme domain and mammalian cytochrome b5, we have predicted the three-dimensional structure of this domain. This showed that the nitrate reductase heme domain is structurally very similar to cytochrome b5 and it also confirmed that the residue involved in E56 mutation is one of the two heme-binding histidines. The two other mutations (mutants A1 and K21) were found to be, respectively, -1 and +1 frameshift mutations resulting in the appearance of an opal stop codon. These sequence data confirmed previous genetic and biochemical hypotheses on nitrate reductase-deficient mutants. Northern blot analysis of these mutants indicated that mutant E56 overexpressed the nitrate reductase mRNA, whereas the nonsense mutations present in the other mutants led to reduced levels of nitrate reductase mRNA.
Meyer
C
C
Laboratoire de Biologie Cellulaire, Institut National de la Recherche Agronomique, Versailles, France.
Levin
J M
JM
Roussel
J M
JM
Rouzé
P
P
D000595:Amino Acid Sequence; D001483:Base Sequence; D015152:Blotting, Northern; D015786:Cytochromes b5 / Q000235:genetics; D004247:DNA / Q000235:genetics; D006418:Heme / Q000235:genetics*; D008969:Molecular Sequence Data; D009154:Mutation; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics*; D010947:Plants, Toxic; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D012313:RNA / Q000235:genetics; D013057:Spectrum Analysis; D014026:Nicotiana / Q000235:genetics*
D006418:Heme; D012313:RNA; D004247:DNA; D015786:Cytochromes b5; D009565:Nitrate Reductases; D050901:Nitrate Reductase