Basis of arginine sensitivity of microbial N-acetyl-L-glutamate kinases:: Mutagenesis and protein engineering study with the Pseudomonas aeruginosa and Escherichia coli enzymes
Basis of arginine sensitivity of microbial N-acetyl-L-glutamate kinases:: Mutagenesis and protein engineering study with the Pseudomonas aeruginosa and Escherichia coli enzymes
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DOI：
10.1128/jb.01831-07
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发表时间：
2008-04-01
影响因子：
3.2
通讯作者：
Rubio, Vicente
中科院分区：
文献类型：
--
作者：
Fernandez-Murga, M. Leonor;Rubio, Vicente
N-乙酰谷氨酸激酶 (NAGK) 催化精氨酸生物合成的第二步。在铜绿假单胞菌中，但在大肠杆菌中则不然，该步骤是限速和反馈，并受到精氨酸的 S 形抑制。晶体结构表明，对精氨酸不敏感的大肠杆菌 NAGK (EcNAGK) 是同二聚体，而对精氨酸抑制的 NAGK，包括铜绿假单胞菌 NAGK (PaNAGK)，是六聚体，其中额外的 N 末端扭结螺旋 (N-helix) 将三个二聚体互连。通过在 PaNAGK 中引入单个氨基酸替换，我们证明了结构鉴定的精氨酸位点的功能，因为精氨酸位点突变选择性地降低了对精氨酸的表观亲和力。影响 R24 和 E17 的 N 螺旋突变分别增加和降低 PaNAGK 对精氨酸的表观亲和力，正如根据酶结构预测的那样，这些酶结构揭示了这些键残基分别形成有利于难以接近和可接近的精氨酸位点构象。 N 螺旋 N 末端缺失跨越 >= 16 个残基，将 PaNAGK 解离为活性二聚体，即
N-Acetylglutamate kinase (NAGK) catalyzes the second step of arginine biosynthesis. In Pseudomonas aeruginosa, but not in Escherichia coli, this step is rate limiting and feedback and sigmoidally inhibited by arginine. Crystal structures revealed that arginine-insensitive E. coli NAGK (EcNAGK) is homodimeric, whereas arginine-inhibitable NAGKs, including P. aeruginosa NAGK (PaNAGK), are hexamers in which an extra N-terminal kinked helix (N-helix) interlinks three dimers. By introducing single amino acid replacements in PaNAGK, we prove the functionality of the structurally identified arginine site, as arginine site mutations selectively decreased the apparent affinity for arginine. N-helix mutations affecting R24 and E17 increased and decreased, respectively, the apparent affinity of PaNAGK for arginine, as predicted from enzyme structures that revealed the respective formation by these residues of bonds favoring inaccessible and accessible arginine site conformations. N-helix N-terminal deletions spanning >= 16 residues dissociated PaNAGK to active dimers, those of