Kinetic analysis of 14-3-3-inhibited arabidopsis thaliana nitrate reductase.
- Author(s)
- Iris Lambeck, Jean-Chih Chi, Sabrina Krizowski, Stefan Mueller, Norbert Mehlmer, Markus Teige, Katrin Fischer, Guenter Schwarz
- 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.
- Organisation(s)
- Department of Biochemistry and Cell Biology
- External organisation(s)
- Universität zu Köln
- Journal
- Biochemistry
- Pages
- 8177-8186
- No. of pages
- 10
- ISSN
- 0006-2960
- Publication date
- 2010
- Peer reviewed
- Yes
- Austrian Fields of Science 2012
- 1060 Biology, 1040 Chemistry
- Portal url
- https://ucris.univie.ac.at/portal/en/publications/kinetic-analysis-of-1433inhibited-arabidopsis-thaliana-nitrate-reductase(2fb07e4b-03df-4701-b529-00b066e07528).html