Biochemical and mutational analysis of glutamine synthetase type III from the rumen anaerobe Ruminococcus albus 8
Biochemical and mutational analysis of glutamine synthetase type III from the rumen anaerobe Ruminococcus albus 8
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DOI：
10.1128/jb.187.21.7481-7491.2005
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发表时间：
2005-11-01
影响因子：
3.2
通讯作者：
Cann, IKO
中科院分区：
文献类型：
--
作者：
Amaya, KR;Kocherginskaya, SA;Cann, IKO
在白纹伊蚊基因组序列中发现了编码谷氨酰胺合成酶I型(GSI)和GSIII的两个不同的基因。GSIII蛋白的同源性通过其相关保守基序的存在而得到证实。克隆了编码GSIII的glnN基因，并在大肠杆菌BL21中进行了表达。对重组蛋白进行了纯化，并进行了生化和物理分析。亚基组织表明，溶液中的蛋白质既以单体形式存在，也以寡聚体形式存在。使用正向和伽马-谷氨酰转移酶(γ-GT)分析进行了动力学研究。在四个GSIII基序中，将保守的谷氨酸残基改变为丙氨酸的突变导致两种方法的GS活性急剧下降，但E380A突变除外，这反而导致正向试验中的活性比野生型蛋白高。通过分别突变位于丙氨酸核苷酸结合位点的两个赖氨酸(K308和K318)，也显示了GSIII活性的降低。最重要的是，在氨限制条件下生长的白曲霉8细胞中存在glnN基因的mRNA转录本，而在氨充足条件下生长的细胞中几乎没有检测到转录本，这表明GSIII在白曲霉的氮代谢中起着重要的作用。此外，对保守的GSIII基序的突变研究首次证明了它们在GSIII蛋白的结构和/或功能中的重要性。
Two different genes encoding glutamine synthetase type I (GSI) and GSIII were identified in the genome sequence of R. albus 8. The identity of the GSIII protein was confirmed by the presence of its associated conserved motifs. The glnN gene, encoding the GSIII, was cloned and expressed in Escherichia coli BL21 cells. The recombinant protein was purified and subjected to biochemical and physical analyses. Subunit organization suggested a protein present in solution as both monomers and oligomers. Kinetic studies using the forward and the gamma-glutamyl transferase (gamma-GT) assays were carried out. Mutations that changed conserved glutamic acid residues to alanine in the four GSIII motifs resulted in drastic decreases in GS activity using both assays, except for an E380A mutation, which rather resulted in an increase in activity in the forward assay compared to the wild-type protein. Reduced GSIII activity was also exhibited by mutating, individually, two lysines (K308 and K318) located in the putative nucleotide-binding site to alanine. Most importantly, the presence of mRNA transcripts of the glnN gene in R. albus 8 cells grown under ammonia limiting conditions, whereas little or no transcript was detected in cells grown under ammonia sufficient conditions, suggested an important role for the GSIII in the nitrogen metabolism of R. albus 8. Furthermore, the mutational studies on the conserved GSIII motifs demonstrated, for the first time, their importance in the structure and/or function of a GSIII protein.