15N Tracing Studies on In Vitro Reactions of Ferredoxin-Dependent Nitrite Reductase and Glutamate Synthase Using Reconstituted Electron Donation Systems.
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.
Department of Applied Biological Chemistry, the University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan tadakatsu_yoneyama@opal.ocn.ne.jp.
Tadakatsu
T
Yoneyama
Department of Applied Biological Chemistry, the University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan.
Tamaki
T
Fujimori
Department of Applied Biological Chemistry, the University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo, 113-8657 Japan.
Shuichi
S
Yanagisawa
Laboratory of Regulation of Biological Reaction, Institute for Protein Research, Osaka University, Suita, Osaka, 565-0871 Japan.
Toshiharu
T
Hase
INRA, Institut Jean-Pierre Bourgin, UMR1318, ERL CNRS 3559, Saclay Plant Sciences, RD10, F-78026 Versailles, France.
Akira
A
Suzuki
D064751:Ammonium Compounds / Q000378:metabolism; D004583:Electrons; D050900:Ferredoxin-Nitrite Reductase / Q000378:metabolism*; D005970:Glutamate Synthase / Q000378:metabolism*; D005971:Glutamates / Q000096:biosynthesis; D018698:Glutamic Acid / Q000378:metabolism; D005972:Glutaminase / Q000378:metabolism; D007553:Isotope Labeling; D009249:NADP / Q000378:metabolism; D009573:Nitrites / Q000378:metabolism; D009587:Nitrogen Isotopes; D011995:Recombination, Genetic / Q000235:genetics; D003313:Zea mays / Q000201:enzymology*
D064751:Ammonium Compounds; D005971:Glutamates; D009573:Nitrites; D009587:Nitrogen Isotopes; D018698:Glutamic Acid; D009249:NADP; D005970:Glutamate Synthase; D050900:Ferredoxin-Nitrite Reductase; D005972:Glutaminase
15N tracing; Electron donation; Glutamate synthase (GOGAT); Nitrite reductase (NiR); Recombinant enzyme; Reconstituted system