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.

Molecular Mechanism of 14-3-3 Protein-mediated Inhibition of Plant Nitrate Reductase

“…The behavior of this fragment was neither viscosity-dependent ( Fig. 3C and supplemental Table S1) nor affected by phosphorylation of Ser-534 and complexation with 14-3-3, indicating that neither the initial reduction of the FAD site by NADH nor the subsequent transfer of reducing equivalents from the FAD to the heme site were affected by phosphorylation and binding of 14-3-3, consistent with previous findings using the holo-enzyme (18). Nevertheless, to more fully characterize the heme-FAD fragment, we examined its reaction with saturating NADH (200 M) by stopped-flow.…”

“…Proteincontaining fractions were concentrated and applied onto a Superdex 200 16/60 column (GE Healthcare) equilibrated in 10 mM Tris/HCl, pH 8.0, 250 mM NaCl. Wild-type Arabidopsis NR was recombinantly expressed in Pichia pastoris KM71, and the calcium-dependent protein kinase CPK-17 as well as the inhibitor protein 14-3-3 were expressed in E. coli and purified as described earlier (18).…”

“…Using a fully defined in vitro expression system for NR from Arabidopsis thaliana, which enabled us to obtain the enzyme in adequate amounts and purity to examine its kinetic properties, we have recently shown that steps 3 or 4, individually or in combination, are the points of 14-3-3-mediated inhibition (18). Of the several 14-3-3 proteins examined, inhibition was found to be most effective using 14-3-3 (18).…”

Reactive Oxygen Species-Dependent Nitric Oxide Production Contributes to Hydrogen-Promoted Stomatal Closure in Arabidopsis

“…Altogether, the above-mentioned data provided genetic evidence that NR is localized downstream of HRW-induced ROS production, which was partially generated by RbohF, leading to NO production and, thereafter, stomatal closure. Certainly, it is worth determining whether NIA1 or NIA2 mediates HRW-activated NO generation in guard cells, and the protein-protein interaction of NR proteins (Kanamaru et al, 1999;Lambeck et al, 2010) (Ohsawa et al, 2007). In this report, however, we found that the HRW stimulated RbohF-dependent ROS synthesis, thereby activating NR-associated NO production, both of which causally resulted in stomatal closure (Figs.…”

Cross-talk of calcium-dependent protein kinase and MAP kinase signaling

“…For example, AtCPK32, a regulator of ABA-responsive gene expression, is localized in the nucleus and at the plasma membrane 28 and AtCPK3, which is involved in salt stress acclimation, is localized at the plasma and the vacuolar membrane, and also in the nucleus and the cytosol. 13,15 Consistently, potential CPK3 targets are localized in the cytosol like nitrate reductase or a phospholipase A 29,30 and at the plasma membrane and the vacuole. 15 In the cytosol both CDPKs and MAPKs have been shown to regulate metabolic enzymes by direct phosphorylation.…”