Yanagisawa T, Lee J T, Wu H C, Kawakami M
Department of Molecular Biology, School of Science, Nagoya University, Japan.
J Biol Chem. 1994 Sep 30;269(39):24304-9.
To elucidate the mode of action of pseudomonic acid, we have compared the deduced amino acid sequences of isoleucyl-tRNA synthetases (ILeRS) from wild-type Escherichia coli strain MC4100, a pseudomonic acid-resistant mutant (strain PS102) of MC4100, and a pseudomonic acid-producing strain, Pseudomonas fluorescens. Compared with the wild-type enzyme, the deduced amino acid sequence of E. coli mutant ileS gene in strain PS102 shows a single amino acid substitution of leucine for phenylalanine at residue 594 of the IleRS. This mutational alteration in IleRS of an E. coli pseudomonic acid-resistant mutant resides in a region of the enzyme in close proximity to one of the consensus sequences of class I aminoacyl-tRNA synthetases, the KMSKS sequence between residues 602 and 606 of the E. coli IleRS. DNA sequence of the cloned ileS gene predicts that the P. fluorescens IleRS consists of 943 amino acids with 54% identity with the E. coli IleRS. The P. fluorescens ileS gene and the wild type and PS102 alleles of E. coli ileS were cloned into an expression vector, pEXPCR, and the sensitivities of E. coli DH5 alpha cells harboring each of these plasmids were compared. The cells harboring the P. fluorescens ileS were found to be most resistant to pseudomonic acid, while the transformants expressing the PS102 IleRS were more resistant than those containing the wild-type E. coli IleRS. IleRS purified from the wild-type E. coli was specifically cleaved by trypsin between Lys605 and Ser606 in the region of K602MSKS606. The protection of the IleRS from the trypsin digestion was found with pseudomonic acid or ATP, but not with isoleucine or tRNA(1Ile). Based on these results, we propose that pseudomonic acid binds to IleRS in the vicinity of the KMSKS sequence that is an ATP-binding subsite, and that pseudomonic acid is a bifunctional inhibitor with characteristics of both isoleucine and ATP, for example, an analog of isoleucyladenylate.
为阐明假单胞菌酸的作用模式，我们比较了野生型大肠杆菌MC4100、MC4100的假单胞菌酸抗性突变株（PS102菌株）和假单胞菌酸产生菌株荧光假单胞菌的异亮氨酰 - tRNA合成酶（ILeRS）推导的氨基酸序列。与野生型酶相比，PS102菌株中大肠杆菌突变ileS基因推导的氨基酸序列在IleRS的第594位残基处显示亮氨酸取代苯丙氨酸的单个氨基酸替换。大肠杆菌假单胞菌酸抗性突变株IleRS中的这种突变改变位于该酶靠近I类氨酰 - tRNA合成酶共有序列之一的区域，即大肠杆菌IleRS第602至606位残基之间的KMSKS序列。克隆的ileS基因的DNA序列预测荧光假单胞菌IleRS由943个氨基酸组成，与大肠杆菌IleRS有54%的同一性。将荧光假单胞菌ileS基因以及大肠杆菌ileS的野生型和PS102等位基因克隆到表达载体pEXPCR中，并比较了携带这些质粒的大肠杆菌DH5α细胞的敏感性。发现携带荧光假单胞菌ileS的细胞对假单胞菌酸最具抗性，而表达PS102 IleRS的转化体比含有野生型大肠杆菌IleRS的转化体更具抗性。从野生型大肠杆菌纯化的IleRS在K602MSKS606区域的Lys605和Ser606之间被胰蛋白酶特异性切割。发现假单胞菌酸或ATP可保护IleRS不被胰蛋白酶消化，但异亮氨酸或tRNA(1Ile)则不能。基于这些结果，我们提出假单胞菌酸在作为ATP结合亚位点的KMSKS序列附近与IleRS结合，并且假单胞菌酸是一种具有异亮氨酸和ATP特征的双功能抑制剂，例如异亮氨酰腺苷酸类似物。
