Universität zu Köln

Lambeck, Iris, Chi, Jen-Chih, Krizowski, Sabina, Mueller, Stefan, Mehlmer, Norbert ORCID: 0000-0002-6854-4341, Teige, Markus ORCID: 0000-0001-7204-1379, Fischer, Katrin and Schwarz, Guenter ORCID: 0000-0002-2118-9338 (2010). Kinetic Analysis of 14-3-3-Inhibited Arabidopsis thaliana Nitrate Reductase. Biochemistry, 49 (37). S. 8177 - 8187. WASHINGTON: AMER CHEMICAL SOC. ISSN 0006-2960

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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 Arahidopsis 14-3-3 proteins, isoforms omega, kappa, and lambda exhibited the strongest inhibition of NR. The kinetic parameters of noninhibited, phosphorylated NR (pNR) and pNR 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 mu M) 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.

Item Type:	Journal Article
Creators:	Creators Email ORCID ORCID Put Code Lambeck, Iris UNSPECIFIED UNSPECIFIED UNSPECIFIED Chi, Jen-Chih UNSPECIFIED UNSPECIFIED UNSPECIFIED Krizowski, Sabina UNSPECIFIED UNSPECIFIED UNSPECIFIED Mueller, Stefan UNSPECIFIED UNSPECIFIED UNSPECIFIED Mehlmer, Norbert UNSPECIFIED orcid.org/0000-0002-6854-4341 UNSPECIFIED Teige, Markus UNSPECIFIED orcid.org/0000-0001-7204-1379 UNSPECIFIED Fischer, Katrin UNSPECIFIED UNSPECIFIED UNSPECIFIED Schwarz, Guenter UNSPECIFIED orcid.org/0000-0002-2118-9338 UNSPECIFIED
URN:	urn:nbn:de:hbz:38-496473
DOI:	10.1021/bi1003487
Journal or Publication Title:	Biochemistry
Volume:	49
Number:	37
Page Range:	S. 8177 - 8187
Date:	2010
Publisher:	AMER CHEMICAL SOC
Place of Publication:	WASHINGTON
ISSN:	0006-2960
Language:	English
Faculty:	Unspecified
Divisions:	Unspecified
Subjects:	no entry
Uncontrolled Keywords:	Keywords Language REGULATORY PHOSPHORYLATION SITE; SUCROSE-PHOSPHATE SYNTHASE; DEPENDENT PROTEIN-KINASE; POSTTRANSLATIONAL REGULATION; CRYSTAL-STRUCTURES; INHIBITOR PROTEIN; OLERACEA LEAVES; 14-3-3 ISOFORMS; IN-VITRO; BINDING Multiple languages Biochemistry & Molecular Biology Multiple languages
URI:	http://kups.ub.uni-koeln.de/id/eprint/49647