Cloning of higher plant glutamate synthase genes

Date

1993-08

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Publisher

Texas Tech University

Abstract

In all plants, glutamate synthase (GOGAT) is a key enzyme involved in the assimilation of ammonia. GOGAT catalyzes the transamidation of the amido nitrogen from glutamine to 2-oxoglutarate to form two molecules of glutamate. GOGAT exists in higher plants in two forms, each of which requires a specific electron donor for the reaction; one utilizes ferredoxin as a reductant and the other utilizes NAD(P)H. Fd-dependent GOGAT, rather than the NAD(P)H form, is the major component in green tissues and is localized in the chloroplast. Fddependent GOGAT from green leaves is a monomeric protein with a molecular mass ranging from 160 to 140 kDa and contains an iron-sulfur cluster and flavins (FAD and FMN).

In this study, I have focused on the cDNA cloning of spinach Fddependent GOGAT in order to elucidate the complete primary structure of the protein and obtain a nucleic acid probe for further genetic studies. The 3.8 kb cDNA insert was isolated from a cDNA library. An additional 1.2 kb coding cDNA fragment was produced by PCR. The total cDNA is 4948 bp in length, and it includes 98% of the coding sequence of the 1483 amino acids of the mature protein. The deduced amino acid sequences reveal a high similarity with Fd-dependent GOGAT from maize and with NADPH-dependent GOGAT from A. brasilense and £. coli. Binding domains for flavin cofactors and ferredoxin, a domain for glutamine binding and activation, and cysteine clusters for iron-sulfur centers formation were tentatively identified on the basis of sequence comparison with flavoproteins, ferredoxin-dependent enzymes, aminotransferases, and iron-sulfur proteins. The cDNA hybridizes to an RNA band of about 5.5 kb. Analysis of genomic DNA indicates the presence of a single-copy gene for Fd-dependent GOGAT in spinach.

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