Abstract
Nitrate reductase (NR) is important for higher land plants, as it catalyzes the rate-limiting step in the nitrate assimilation pathway, the two-electron reduction of nitrate to nitrite. Furthermore, it is considered to be a major enzymatic source of the important signaling molecule nitric oxide (NO), that is produced in a one-electron reduction of nitrite. Like many other plants, the model plant Arabidopsis thaliana expresses two isoforms of NR (NIA1 and NIA2). Up to now, only NIA2 has been the focus of detailed biochemical studies, while NIA1 awaits biochemical characterization. In this study, we have expressed and purified functional fragments of NIA1 and subjected them to various biochemical assays for comparison with the corresponding NIA2-fragments. We analyzed the kinetic parameters in multiple steady-state assays using nitrate or nitrite as substrate and measured either substrate consumption (nitrate or nitrite) or product formation (NO). Our results show that NIA1 is the more efficient nitrite reductase while NIA2 exhibits higher nitrate reductase activity, which supports the hypothesis that the isoforms have special functions in the plant. Furthermore, we successfully restored the physiological electron transfer pathway of NR using reduced nicotinamide adenine dinucleotide (NADH) and nitrate or nitrite as substrates by mixing the N-and C-terminal fragments of NR, thus, opening up new possibilities to study NR activity, regulation and structure.
Highlights
In higher land plants, nitrate is the preferred nutrient for the nitrogen (N) assimilation pathway [1,2].Nitrate reductase (NR, EC 1.7.1.1), which catalyzes the first intracellular and rate-limiting step in nitrate assimilation, is a homodimer of two approximately 100 kDa polypeptide chains, each of which binds three cofactors in individually folded domains
We produced functional proteins of the two NR isoforms from A. thaliana and subjected them to steady-state enzymatic studies to characterize their functional properties. We found that both isoforms are able to use either nitrate or nitrite as a substrate, with NIA2 having a clear preference for nitrate reductase activity, while NIA1 is the more efficient nitrite reductase, and the nitrite reducing activities of both were inhibited at low concentrations of nitrate
We have shown in the past that the N-terminal fragment of Arabidopsis thaliana
Summary
Nitrate is the preferred nutrient for the nitrogen (N) assimilation pathway [1,2]. Nitrate reductase (NR, EC 1.7.1.1), which catalyzes the first intracellular and rate-limiting step in nitrate assimilation, is a homodimer of two approximately 100 kDa polypeptide chains, each of which binds three cofactors in individually folded domains. The C-terminal domain carrying a flavine adenine dinucleotide (FAD) cofactor accepts two electrons from NADH or the phosphorylated form NADPH and passes them sequentially to the middle domain containing a b5 -type cytochrome heme. The electrons are shuttled to the molybdenum cofactor (Moco)-containing catalytic site in the N-terminal domain, and it is here that substrate reduction takes place. Three non-conserved flexible regions are found in NR: an N-terminal peptide preceding the Moco-domain, and two linkers connecting the central heme-domain to the Moco-domain (hinge 1)
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
