Glutamate synthases from conifers: gene structure and phylogenetic studies.

BACKGROUND: Plants synthesize glutamate from ammonium by the combined activity of the enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) through the glutamate synthase cycle. In plants, there are two forms of glutamate synthases that differ in their electron donors, NADH-GOGAT (EC 1.4.1.14) and Fd-GOGAT (EC 1.4.7.1), which have differential roles either in primary ammonia assimilation or in the reassimilation of ammonium from different catabolic processes. Glutamate synthases are complex iron-sulfur flavoproteins containing functional domains involved in the control and coordination of their catalytic activities in annual plants. In conifers, partial cDNA sequences for GOGATs have been isolated and used for gene expression studies. However, knowledge of the gene structure and of phylogenetic relationships with other plant enzymes is quite scant. RESULTS: Technological advances in conifer megagenomes sequencing have made it possible to obtain full-length cDNA sequences encoding Fd- and NADH-GOGAT from maritime pine, as well as BAC clones containing sequences for NADH-GOGAT and Fd-GOGAT genes. In the current study, we studied the genomic organization of pine GOGAT genes, the size of their exons/introns, copy numbers in the pine genome and relationships with other plant genes. Phylogenetic analysis was performed, and the degree of preservation and dissimilarity of key domains for the catalytic activities of these enzymes in different taxa were determined. CONCLUSIONS: Fd- and NADH-GOGAT are encoded by single-copy genes in the maritime pine genome. The Fd-GOGAT gene is extremely large spanning more than 330 kb and the presence of very long introns highlights the important contribution of LTR retrotransposons to the gene size in conifers. In contrast, the structure of the NADH-GOGAT gene is similar to the orthologous genes in angiosperms. Our phylogenetic analysis indicates that these two genes had different origins during plant evolution. The results provide new insights into the structure and molecular evolution of these essential genes.

Protection of the glutamate pool concentration in enteric bacteria.

The central nitrogen metabolic circuit in enteric bacteria consists of three enzymes: glutamine synthetase, glutamate synthase (GOGAT), and glutamate dehydrogenase (GDH). With the carbon skeleton provided by 2-oxoglutarate, ammonia/ammonium (NH(4)(+)) is assimilated into two central nitrogen intermediates, glutamate and glutamine. Although both serve as nitrogen donors for all biosynthetic needs, glutamate and glutamine play different roles. Internal glutamine serves as a sensor of external nitrogen availability, and its pool concentration decreases upon nitrogen limitation. A high glutamate pool concentration is required to maintain the internal K(+) pool. The configuration of high glutamate and low glutamine pools was disrupted in GOGAT(-) mutants under low NH(4)(+) conditions: the glutamate pool was low, the difference between glutamate and glutamine was diminished, and growth was defective. When a GOGAT(-) mutant was cultured in an NH(4)(+)-limited chemostat, two sequential spontaneous mutations occurred. Each resulted in a suppressor mutant that outgrew its predecessor in the chemostat. The first suppressor overexpressed GDH, and the second also had a partially impaired glutamine synthetase. The result was a triple mutant in which NH(4)(+) was assimilated by two enzymes instead of the normal three and yet glutamate and glutamine pools and growth were essentially normal. The results indicate preference for the usual ratio of glutamate and glutamine and the resilient and compensatory nature of the circuit on pool control. Analysis of other suppressor mutants selected on solid medium suggests that increased GDH expression is the key for rescue of the growth defect of GOGAT(-) mutants under low NH(4)(+) conditions.