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15N Tracing Studies on In Vitro Reactions of Ferredoxin-Dependent Nitrite Reductase and Glutamate Synthase Using Reconstituted Electron Donation Systems.
- Source :
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Plant & cell physiology [Plant Cell Physiol] 2015 Jun; Vol. 56 (6), pp. 1154-61. Date of Electronic Publication: 2015 Mar 05. - Publication Year :
- 2015
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Abstract
- It is known that plants contain ferredoxin (Fd)-dependent nitrite reductase (NiR) and glutamate synthase (GOGAT). The Fd-NiR reaction produces ammonia from nitrite, and the activity is usually measured by nitrite disappearance. The Fd-GOGAT reaction forms two glutamates of different origin, from glutamine and 2-oxoglutarate, and the activity is measured by the oxidation of reductant (NADPH) or by formation of total glutamate. Here, a quantitative probe of the products and efficiency of the process was conducted using (15)N tracing techniques on these reactions in vitro. We quantified the reduction of (15)N-labeled [Formula: see text] to [Formula: see text] and the formation of [(15)N]glutamate and [(14)N]glutamate from [5-(15)N-amide]glutamine plus 2-oxoglutarate by NiR and GOGAT, respectively, with the reductant-Fd-NADP(+) oxidoreductase (FNR)-Fd system as the sequential electron donors. The supply of dithionite or NADPH to recombinant cyanobacterial NiR led to electron donation system-dependent formation of [(15)N]ammonium from [(15)N]nitrite. Addition of 20 mM NaCl and 20 mM Na-ascorbate accelerated nitrite reduction under high concentrations of NADPH. A sufficient supply of NADPH to recombinant Zea mays Fd-GOGAT generated complete GOGAT activity (transferring the [5-(15)N]amide of glutamine to 2-oxoglutarate to form [(15)N]glutamate), whereas a shortage of NADPH resulted in glutaminase activity only, which removed the amide from glutamine and released ammonia and [(14)N]glutamate. We conclude that although the recombinant Fd-GOGAT enzyme has two forms of glutamate synthesis, the first by glutaminase (ammonia release by glutamine amidotransferase) and the second by glutamate synthase (coupling of the ammonia and exogenously applied 2-oxoglutarate), the first works without NADPH, while the second is strictly dependent on NADPH availability.<br /> (© The Author 2015. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.)
- Subjects :
- Ammonium Compounds metabolism
Glutamates biosynthesis
Glutamic Acid metabolism
Glutaminase metabolism
NADP metabolism
Nitrites metabolism
Nitrogen Isotopes
Recombination, Genetic genetics
Electrons
Ferredoxin-Nitrite Reductase metabolism
Glutamate Synthase metabolism
Isotope Labeling
Zea mays enzymology
Subjects
Details
- Language :
- English
- ISSN :
- 1471-9053
- Volume :
- 56
- Issue :
- 6
- Database :
- MEDLINE
- Journal :
- Plant & cell physiology
- Publication Type :
- Academic Journal
- Accession number :
- 25745028
- Full Text :
- https://doi.org/10.1093/pcp/pcv039