Akochy Pierre-Marie, Bernard Dominic, Roy Paul H, Lapointe Jacques
Département de Biochimie et de Microbiologie, Faculté des Sciences et de Génie, Université Laval, Québec, Canada G1K 7P4.
J Bacteriol. 2004 Feb;186(3):767-76. doi: 10.1128/JB.186.3.767-776.2004.
The genomic sequence of Pseudomonas aeruginosa PAO1 was searched for the presence of open reading frames (ORFs) encoding enzymes potentially involved in the formation of Gln-tRNA and of Asn-tRNA. We found ORFs similar to known glutamyl-tRNA synthetases (GluRS), glutaminyl-tRNA synthetases (GlnRS), aspartyl-tRNA synthetases (AspRS), and trimeric tRNA-dependent amidotransferases (AdT) but none similar to known asparaginyl-tRNA synthetases (AsnRS). The absence of AsnRS was confirmed by biochemical tests with crude and fractionated extracts of P. aeruginosa PAO1, with the homologous tRNA as the substrate. The characterization of GluRS, AspRS, and AdT overproduced from their cloned genes in P. aeruginosa and purified to homogeneity revealed that GluRS is discriminating in the sense that it does not glutamylate tRNA(Gln), that AspRS is nondiscriminating, and that its Asp-tRNA(Asn) product is transamidated by AdT. On the other hand, tRNA(Gln) is directly glutaminylated by GlnRS. These results show that P. aeruginosa PAO1 is the first organism known to synthesize Asn-tRNA via the indirect pathway and to synthesize Gln-tRNA via the direct pathway. The essential role of AdT in the formation of Asn-tRNA in P. aeruginosa and the absence of a similar activity in the cytoplasm of eukaryotic cells identifies AdT as a potential target for antibiotics to be designed against this human pathogen. Such novel antibiotics could be active against other multidrug-resistant gram-negative pathogens such as Burkholderia and Neisseria as well as all pathogenic gram-positive bacteria.
对铜绿假单胞菌PAO1的基因组序列进行搜索，以寻找编码可能参与谷氨酰胺-tRNA和天冬酰胺-tRNA形成的酶的开放阅读框（ORF）。我们发现了与已知的谷氨酰-tRNA合成酶（GluRS）、谷氨酰胺-tRNA合成酶（GlnRS）、天冬氨酰-tRNA合成酶（AspRS）和三聚体tRNA依赖性酰胺转移酶（AdT）相似的ORF，但没有与已知的天冬酰胺-tRNA合成酶（AsnRS）相似的。以同源tRNA为底物，通过对铜绿假单胞菌PAO1的粗提物和分级提取物进行生化测试，证实了AsnRS的缺失。对从其在铜绿假单胞菌中克隆的基因过量表达并纯化至均一的GluRS、AspRS和AdT的表征表明，GluRS具有区分性，即它不会使tRNA(Gln)谷氨酰化，AspRS不具有区分性，其Asp-tRNA(Asn)产物被AdT转酰胺化。另一方面，tRNA(Gln)被GlnRS直接谷氨酰胺化。这些结果表明，铜绿假单胞菌PAO1是已知的第一种通过间接途径合成天冬酰胺-tRNA并通过直接途径合成谷氨酰胺-tRNA的生物体。AdT在铜绿假单胞菌中天冬酰胺-tRNA形成中的重要作用以及真核细胞细胞质中缺乏类似活性，确定AdT是针对这种人类病原体设计的抗生素的潜在靶点。这种新型抗生素可能对其他多重耐药革兰氏阴性病原体如伯克霍尔德菌和奈瑟菌以及所有致病性革兰氏阳性细菌具有活性。
