Purification and characterization of a new type of glutamine synthetase from cyanobacteria.
The cyanobacterium Synechocystis sp. PCC 6803 contains two genes encoding two different types of glutamine synthetases (GS), glnA and glnN. The first codes for a typical prokaryotic GS type I and the second one codes for a GS type III, different in amino acid sequence to the prokaryotic GSI and the eukaryotic GSII. The glnN gene has been expressed in Escherichia coli and the corresponding protein purified almost to homogeneity (92%). The native enzyme (500 kDa) was composed of six identical subunits with an apparent molecular mass of 80 kDa. The protein was strongly stabilized in the presence of Mn2+ but not with other divalent cations. Biosynthetic activity of GSIII required the same substrates and cofactors as GSI and GSII enzymes. Apparent Km values for ATP, glutamate and ammonium were 0.43 mM, 0.9 mM and 0.19 mM, respectively. The enzyme was weakly inhibited by several amino acids and strongly inhibited by ADP. Synechocystis GSIII was also inhibited by L-methionine sulfoximine and DL-phosphinotricin, two transition-state analogs of the GS reaction mechanism. GSIII has also been purified from nitrogen-starved Synechocystis 6803 glnA mutant cells, demonstrating that the GS activity, strongly induced under nitrogen starvation in these cells, corresponds to the glnN gene product. In addition, a Synechocystis 6803 glnN mutant lacks the corresponding 80-kDa protein (GSIII). Polyclonal antibodies specific for GSIII cross-react with GSIII from other cyanobacteria. In all the strains analysed, levels of GSIII protein increased under nitrogen deficiency. These data suggest that GSIII is specifically required under conditions of nitrogen starvation.
García-Domínguez
M
M
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Spain.
Reyes
J C
JC
Florencio
F J
FJ
D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification; D000458:Cyanobacteria / Q000201:enzymology* / Q000254:growth & development / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005798:Genes, Bacterial; D005822:Genetic Vectors; D005974:Glutamate-Ammonia Ligase / Q000096:biosynthesis / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification*; D007700:Kinetics; D009154:Mutation; D009584:Nitrogen / Q000172:deficiency
D001426:Bacterial Proteins; D005974:Glutamate-Ammonia Ligase; D009584:Nitrogen