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SYSTEMS BIOLOGY, MOLECULAR BIOLOGY, AND GENE REGULATION

The Isoenzyme 7 of Tobacco NAD(H)-Dependent Glutamate Dehydrogenase Exhibits High Deaminating and Low Aminating Activities in Vivo^1,[OA]

Department of Biology (D.S.S., N.V.P., K.A.R.-A.) and Department of Chemistry (A.K., A.S., E.G.S.), University of Crete, 71409 Heraklion, Greece

Following the discovery of glutamine synthetase/glutamate (Glu) synthase, the physiological roles of Glu dehydrogenase (GDH) in nitrogen metabolism in plants remain obscure and is the subject of considerable controversy. Recently, transgenics were used to overexpress the gene encoding for the β-subunit polypeptide of GDH, resulting in the GDH-isoenzyme 1 deaminating in vivo Glu. In this work, we present transgenic tobacco (Nicotiana tabacum) plants overexpressing the plant gdh gene encoding for the -subunit polypeptide of GDH. The levels of transcript correlated well with the levels of total GDH protein, the -subunit polypeptide, and the abundance of GDH-anionic isoenzymes. Assays of transgenic plant extracts revealed high in vitro aminating and low deaminating activities. However, gas chromatography/mass spectrometry analysis of the metabolic fate of ¹⁵NH₄ or [¹⁵N]Glu revealed that GDH-isoenzyme 7 mostly deaminates Glu and also exhibits low ammonium assimilating activity. These and previous results firmly establish the direction of the reactions catalyzed by the anionic and cationic isoenzymes of GDH in vivo under normal growth conditions and reveal a paradox between the in vitro and in vivo enzyme activities.

The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Kalliopi A. Roubelakis-Angelakis (poproube{at}biology.uoc.gr).

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www.plantphysiol.org/cgi/doi/10.1104/pp.107.107813

^* Corresponding author; e-mail poproube{at}biology.uoc.gr.

Received August 21, 2007; accepted September 24, 2007; published October 11, 2007.

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