García-Domínguez M, Reyes J C, Florencio F J
Instituto de Bioquímica Vegetal y Fotosíntesis, Universidad de Sevilla-CSIC, Spain.
Eur J Biochem. 1997 Feb 15;244(1):258-64. doi: 10.1111/j.1432-1033.1997.00258.x.
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.
集胞藻6803含有两个编码不同类型谷氨酰胺合成酶（GS）的基因，即glnA和glnN。第一个基因编码典型的原核生物I型GS，第二个基因编码III型GS，其氨基酸序列与原核生物GSI和真核生物GSII不同。glnN基因已在大肠杆菌中表达，相应的蛋白质纯化后几乎达到同质（92%）。天然酶（500 kDa）由六个相同的亚基组成，表观分子量为80 kDa。该蛋白质在Mn2+存在下能得到强烈稳定，但在其他二价阳离子存在下则不然。GSIII的生物合成活性所需的底物和辅因子与GSI和GSII酶相同。ATP、谷氨酸和铵的表观Km值分别为0.43 mM、0.9 mM和0.19 mM。该酶受到几种氨基酸的弱抑制，受到ADP的强烈抑制。集胞藻GSIII也受到L-甲硫氨酸亚砜亚胺和DL-草丁膦的抑制，这两种物质是GS反应机制的两种过渡态类似物。GSIII也已从氮饥饿的集胞藻6803 glnA突变细胞中纯化出来，这表明在这些细胞中氮饥饿时强烈诱导的GS活性对应于glnN基因产物。此外，集胞藻6803 glnN突变体缺乏相应的80 kDa蛋白质（GSIII）。针对GSIII的多克隆抗体与其他蓝细菌的GSIII发生交叉反应。在所有分析的菌株中，GSIII蛋白水平在氮缺乏条件下升高。这些数据表明，在氮饥饿条件下特别需要GSIII。
