A glycine to aspartic acid change in the MoCo domain of nitrate reductase reduces both activity and phosphorylation levels in Arabidopsis.
Nitrate reductase (NR), the first enzyme in the nitrate assimilation pathway, is regulated post-transcriptionally in response to light and CO2. In spinach, it has been shown that phosphorylation is one mechanism that mediates this regulation. In this paper, the phosphorylation of NR in Arabidopsis is described in both wild-type and NR- mutant plants. A 110-kDa protein radiolabeled in vivo with 32PO4 was immunoprecipitated with anti-NR antibody from extracts of wild-type plants but not of mutant plants in which the NR gene NIA2 had been deleted. Phosphoamino acid and phosphopeptide analysis showed that, as for spinach, NR from Arabidopsis is phosphorylated on serine and produces multiple phosphopeptides upon digestion with CNBr or trypsin. Analysis of three mutants with lesions in the NIA2 NR structural gene showed that one mutant, chl3-1, has a reduced phosphorylation phenotype that is not complemented by a NR deletion mutant. Comparison of the sequences of the wild-type and chl3-1 NIA2 genes revealed a single base mutation changing a glycine codon to an aspartic acid codon. This glycine, at position 308 in the MoCo domain of NR, is completely conserved in all known eukaryotic NR sequences. Thus, glycine 308 is required for normal activity and phosphorylation of NR, and substitution of this residue with aspartic acid disrupts both processes, most likely by altering the conformation of the NR MoCo domain.
LaBrie
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Department of Biology, University of California, San Diego, La Jolla 92093-0116.
Crawford
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D000595:Amino Acid Sequence; D000818:Animals; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D001224:Aspartic Acid; D002248:Carbon Monoxide; D002645:Chickens; D017343:Genes, Plant; D005998:Glycine; D008099:Liver / Q000201:enzymology; D008969:Molecular Sequence Data; D008982:Molybdenum; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000096:biosynthesis / Q000235:genetics / Q000378:metabolism*; D050862:Oxidoreductases Acting on Sulfur Group Donors / Q000235:genetics; D010766:Phosphorylation; D017354:Point Mutation; D051381:Rats; D017386:Sequence Homology, Amino Acid
D001224:Aspartic Acid; D002248:Carbon Monoxide; D008982:Molybdenum; D009565:Nitrate Reductases; D050901:Nitrate Reductase; D050862:Oxidoreductases Acting on Sulfur Group Donors; D005998:Glycine