Journal Article

Identification of Ser-543 as the major regulatory phosphorylation site in spinach leaf nitrate reductase

Bachmann, M. (USDA, ARS, North Carolina State University, Raleigh, NC.), Huber, S.C, Yoo, B.C, Harmon, A.C, Campbell, W.H, Shiraishi, N

1996

Overview

Spinach leaf NADH:nitrate reductase (NR) responds to light/dark signals and photosynthetic activity in part as a result of rapid regulation by reversible protein phosphorylation. We have identified the major regulatory phosphorylation site as Ser-543, which is located in the hinge 1 region connecting the cytochrome b domain with the molybdenum-pterin cofactor binding domain of NR, using recombinant NR fragments containing or lacking the phosphorylation site sequence. Studies with NR partial reactions indicated that the block in electron flow caused by phosphorylation also could be localized to the hinge 1 region. A synthetic peptide (NR6) based on the phosphorylation site sequence was phosphorylated readily by NR kinase (NRk) in vitro. NR6 kinase activity tracked the ATP-dependent inactivation of NR during several chromatographic steps and completely inhibited inactivation/phosphorylation of native NR in vitro. Two forms of NRk were resolved by using anion exchange chromatography. Studies with synthetic peptide analogs indicated that both forms of NRk had similar specificity determinants, requiring a basic residue at P-3 (i.e., three amino acids N-terminal to the phosphorylated serine) and a hydrophobic residue at P-5. Both forms are strictly calcium dependent but belong to distinct families of protein kinases because they are distinct immunochemically.

• Publisher: American Society of Plant Physiologists
• Subject: ACTIVIDAD ENZIMATICA / ACTIVITE ENZYMATIQUE / Amino Acid Sequence / amino acid sequences / Amino acids / Binding Sites / chemical synthesis / chemistry / Chromatography / Chromatography, Ion Exchange / COMPOSICION QUIMICA / COMPOSITION CHIMIQUE / Cytochrome b Group / Cytochrome b Group - metabolism / Cytochromes / enzyme activity / Enzymes / enzymology / FOSFORILACION / Genes, Plant / genetics / ISOENZIMAS / ISOENZYME / isolation & purification / isozymes / Kinetics / Life Sciences (General) / metabolism / Molecular Sequence Data / Nitrate Reductase / nitrate reductase (NADH) / Nitrate Reductases / Nitrate Reductases - chemistry / Nitrate Reductases - genetics / Nitrate Reductases - metabolism / Nitrates / OXIDORREDUCTASAS / OXYDOREDUCTASE / PEPTIDE / Peptide Fragments / Peptide Fragments - chemistry / Peptides / Peptides - chemical synthesis / Peptides - chemistry / Peptides - pharmacology / PEPTIDOS / pharmacology / PHOSPHORYLATION / Photosynthesis / Plants / Plants - enzymology / Plants - genetics / PROTEINA QUINASA / PROTEINE KINASE / Proteins / Recombinant Proteins / Recombinant Proteins - chemistry / Recombinant Proteins - isolation & purification / Recombinant Proteins - metabolism / Sequence Homology, Amino Acid / SERINA / SERINE / Soybeans / Spinach / SPINACIA OLERACEA / Spinacia oleracea - enzymology / Spinacia oleracea - genetics / synthetic peptides