Shatters R G, Liu Y, Kahn M L
Department of Genetics and Cell Biology, Washington State University, Pullman 99164-6340.
J Biol Chem. 1993 Jan 5;268(1):469-75.
Two glutamine synthetases, GSI and GSII, are found in most rhizobia. However, WSU650, a Rhizobium meliloti glnA glnII mutant that lacks both enzymes, can grow without a glutamine supplement in minimal medium that contains both ammonium and glutamate. The bacteria contained a third glutamine synthetase, GSIII, which has been purified and partially characterized. GSIII had considerable glutamine synthetase activity when assayed using a semibiosynthetic (glutamate- and hydroxylamine-dependent) assay, but had no detectable transferase (glutamine- and hydroxyl-amine-dependent) activity. GSIII was inhibited by ADP and pyrophosphate but not by various nitrogen-containing metabolites that inhibit other GS enzymes. Activity was also inhibited by methionine sulfoximine, a transition state analog, but the concentration needed to inhibit GSIII was 50 to 100 times higher than that needed to inhibit GSI or GSII. GSIII had a Km for glutamate of 13.3 mM, for ammonium of 33 mM, and for hydroxylamine of 5.3 mM with a pH optimum of 6.8 and a temperature optimum of 50 degrees C. The purified protein had related subunits of 46.5 and 49 kDa and a native molecular mass of 355 kDa, indicating the native enzyme was an octamer. Polyclonal antibodies specific for GSIII reacted with a protein of similar molecular weight in Escherichia coli strains that carry R. meliloti glnT on a plasmid. GSIII activity was detected in some of these strains that contained glnT. Extracts of root nodules formed by WSU650 also react with the antibodies.
在大多数根瘤菌中发现了两种谷氨酰胺合成酶，即GSI和GSII。然而，苜蓿根瘤菌glnA glnII突变体WSU650缺乏这两种酶，在含有铵和谷氨酸的基本培养基中，无需补充谷氨酰胺就能生长。该细菌含有第三种谷氨酰胺合成酶GSIII，已对其进行了纯化和部分特性分析。使用半生物合成法（依赖谷氨酸和羟胺）检测时，GSIII具有相当高的谷氨酰胺合成酶活性，但未检测到转移酶（依赖谷氨酰胺和羟胺）活性。GSIII受到ADP和焦磷酸的抑制，但不受抑制其他GS酶的各种含氮代谢物的抑制。甲硫氨酸亚砜亚胺（一种过渡态类似物）也会抑制其活性，但抑制GSIII所需的浓度比抑制GSI或GSII所需的浓度高50至100倍。GSIII对谷氨酸的Km值为13.3 mM，对铵的Km值为33 mM，对羟胺的Km值为5.3 mM，最适pH为6.8，最适温度为50℃。纯化后的蛋白质具有46.5 kDa和49 kDa的相关亚基，天然分子量为355 kDa，表明天然酶是八聚体。对GSIII具有特异性的多克隆抗体与携带苜蓿根瘤菌glnT质粒的大肠杆菌菌株中分子量相似的蛋白质发生反应。在一些含有glnT的菌株中检测到了GSIII活性。WSU650形成的根瘤提取物也与这些抗体发生反应。
