title: Kinetic analysis of 14-3-3-inhibited Arabidopsis thaliana nitrate reductase.
abstract: Eukaryotic assimilatory nitrate reductase (NR) is a dimeric multidomain molybdo-heme-flavo protein that catalyzes the first and rate-limiting step in the nitrate assimilation of plants, algae, and fungi. Nitrate reduction takes place at the N-terminal molybdenum cofactor-containing domain. Reducing equivalents are derived from NADH, which reduce the C-terminal FAD domain followed by single-electron transfer steps via the middle heme domain to the molybdenum center. In plants, nitrate reduction is post-translationally inhibited by phosphorylation and subsequent binding of 14-3-3 protein to a conserved phosphoserine located in the surface-exposed hinge between the catalytic and heme domain. Here we investigated Arabidopsis thaliana NR activity upon phosphorylation and 14-3-3 binding by using a fully defined in vitro system with purified proteins. We demonstrate that among different calcium-dependent protein kinases (CPKs), CPK-17 efficiently phosphorylates Ser534 in NR. Out of eight purified Arabidopsis 14-3-3 proteins, isoforms ω, κ, and λ exhibited the strongest inhibition of NR. The kinetic parameters of noninhibited, phosphorylated NR (pNR) and pNR in a complex with 14-3-3 were investigated. An 18-fold reduction in k(cat) and a decrease in the apparent K(M)(nitrate) (from 280 to 141 μM) were observed upon binding of 14-3-3 to pNR, suggesting a noncompetitive inhibition with a preferential binding to the substrate-bound state of the enzyme. Recording partial activities of NR demonstrated that the transfer of electrons to the heme is not affected by 14-3-3 binding. The Ser534Ala variant of NR was not inhibited by 14-3-3 proteins. We propose that 14-3-3 binding to Ser534 blocks the transfer of electrons from heme to nitrate by arresting the domain movement via hinge 1.
authors.affiliation: Institute of Biochemistry, Department of Chemistry, University of Cologne, 50674 Cologne, Germany.
authors.forename: Iris
authors.initials: I
authors.lastname: Lambeck
authors.forename: Jen-Chih
authors.initials: JC
authors.lastname: Chi
authors.forename: Sabina
authors.initials: S
authors.lastname: Krizowski
authors.forename: Stefan
authors.initials: S
authors.lastname: Mueller
authors.forename: Norbert
authors.initials: N
authors.lastname: Mehlmer
authors.forename: Markus
authors.initials: M
authors.lastname: Teige
authors.forename: Katrin
authors.initials: K
authors.lastname: Fischer
authors.forename: Guenter
authors.initials: G
authors.lastname: Schwarz
mesh_terms: D048948:14-3-3 Proteins / Q000378:metabolism*; D017360:Arabidopsis / Q000201:enzymology* / Q000378:metabolism; D002384:Catalysis; D003067:Coenzymes; D056890:Eukaryota; D006418:Heme / Q000378:metabolism; D007700:Kinetics; D008667:Metalloproteins; D008982:Molybdenum / Q000378:metabolism; D000090006:Molybdenum Cofactors; D009243:NAD; D050901:Nitrate Reductase / Q000378:metabolism; D050895:Nitrate Reductase (NADH) / Q000378:metabolism*; D009565:Nitrate Reductases / Q000737:chemistry / Q000378:metabolism; D010084:Oxidation-Reduction; D010766:Phosphorylation; D011494:Protein Kinases; D011621:Pteridines; D017871:Calcium-Calmodulin-Dependent Protein Kinases
chemical_list: D048948:14-3-3 Proteins; D003067:Coenzymes; D006418:Heme; D008667:Metalloproteins; D008982:Molybdenum; D000090006:Molybdenum Cofactors; D009243:NAD; D050901:Nitrate Reductase; D050895:Nitrate Reductase (NADH); D009565:Nitrate Reductases; D011494:Protein Kinases; D011621:Pteridines; D017871:Calcium-Calmodulin-Dependent Protein Kinases; C022261:molybdopterin