Jenal U, Rechsteiner T, Tan P Y, Bühlmann E, Meile L, Leisinger T
Mikrobiologisches Institut, Eidgenössische Technische Hochschule, Zürich, Switzerland.
J Biol Chem. 1991 Jun 5;266(16):10570-7.
The ileS gene encoding the isoleucyl-tRNA synthetase of the thermophilic archaebacterium Methanobacterium thermoautotrophicum Marburg was isolated and sequenced. ileS was closely flanked by an unknown open reading frame and by purL and thus is arranged differently from the organizations observed in several eubacteria or in Saccharomyces cerevisiae. The deduced amino acid sequence of isoleucyl-tRNA synthetase was compared with primary sequences of isoleucyl-, valyl-, leucyl-, and methionyl-tRNA synthetases from eubacteria and yeast. The archaebacterial enzyme fitted well into this group of enzymes. It contained the two short consensus sequences observed in class I aminoacyl-tRNA synthetases as well as regions of homology with enzymes of the isoleucine family. Comparison between the isoleucyl-tRNA synthetases of M. thermoautotrophicum yielded 36% amino acid identity with the yeast enzyme and 32% identity with the corresponding enzyme from Escherichia coli. The ileS gene of the pseudomonic acid-resistant M. thermoautotrophicum mutant MBT10 was also sequenced. The mutant enzyme had undergone a glycine to aspartic acid transition at position 590, in a conserved region comprising the KMSKS consensus sequence. The inhibition constants of pseudomonic acid, KiIle and KiATP, for the mutant enzyme were 10-fold higher than those determined for the wild-type enzyme. Both the mutant and the wild-type ileS gene were expressed in E. coli, and their products displayed the expected difference in sensitivity toward pseudomonic acid.
编码嗜热自养甲烷杆菌马尔堡菌株异亮氨酰 - tRNA合成酶的ileS基因被分离并测序。ileS基因紧密毗邻一个未知的开放阅读框以及purL基因，因此其排列方式与在几种真细菌或酿酒酵母中观察到的组织形式不同。将推导得到的异亮氨酰 - tRNA合成酶氨基酸序列与来自真细菌和酵母的异亮氨酰、缬氨酰、亮氨酰和甲硫氨酰 - tRNA合成酶的一级序列进行比较。该古细菌酶很好地归入了这组酶中。它包含在I类氨酰 - tRNA合成酶中观察到的两个短共有序列以及与异亮氨酸家族酶的同源区域。嗜热自养甲烷杆菌的异亮氨酰 - tRNA合成酶与酵母酶的氨基酸同一性为36%，与大肠杆菌相应酶的同一性为32%。对耐假单胞菌酸的嗜热自养甲烷杆菌突变体MBT10的ileS基因也进行了测序。突变酶在包含KMSKS共有序列的保守区域第590位发生了甘氨酸到天冬氨酸的转变。突变酶对假单胞菌酸的抑制常数KiIle和KiATP比野生型酶测定的值高10倍。突变型和野生型ileS基因都在大肠杆菌中表达，它们的产物对假单胞菌酸的敏感性表现出预期的差异。
