Liu J, Gagnon Y, Gauthier J, Furenlid L, L'Heureux P J, Auger M, Nureki O, Yokoyama S, Lapointe J
Département de Biochimie, Faculté des Sciences et de Génie, Université Laval, Ste-Foy, Québec, Canada.
J Biol Chem. 1995 Jun 23;270(25):15162-9. doi: 10.1074/jbc.270.25.15162.
The zinc contents of fragments of Escherichia coli glutamyl-tRNA synthetase, as well as the conservation of the CYC sequence only in zinc-containing glutamyl-tRNA synthetases, suggested that the 98CYCX24-CRHSHEHHADDEPC138 includes some or all residues involved in binding its zinc atom (Liu, J., Lin, S.-X., Blochet, J.-E., Pézolet, M., and Lapointe, J. (1993) Biochemistry 32, 11390-11396). Extended x-ray absorption fine structure (EXAFS) shows that this zinc atom has a four-coordinate non-planar coordination environment with 3 sulfur and 1 nitrogen atoms with bond lengths, respectively, 2.37 +/- 0.02 A and 2.01 +/- 0.02 A, presumably belonging to 3 cysteine residues and 1 histidine residue. Conservative replacement of each histidine and cysteine residue of the 98C-138C segment, respectively, with glutamine (Q) and serine (S), yields variants H129Q, H131Q, H132Q, and C138S (which sustain the growth at 42 degrees C of E. coli JP1449, whose glutamyl-tRNA synthetase is thermosensitive) and C98S, C100S, C125S, and H127Q (which do not). The amount of this enzyme in these mutants is at least 1 order of magnitude larger than that in a wild type strain; however, no glutamyl-tRNA synthetase activity is detectable in extracts of the variants C100S and C125S, whereas its specific activity in those of C98S and H127Q is about 10-fold lower than in cells overproducing the wild type enzyme or the variants H129Q, H131Q, H132Q, and C138S. These results indicate that the zinc atom present in E. coli glutamyl-tRNA synthetase is bound by the 2 evolutionarily conserved cysteines at positions 98 and 100, and by Cys125 and His127. Molecular modeling of the N-terminal half of this enzyme, using the known structure of E. coli glutaminyl-tRNA synthetase, supports this conclusion and suggests that the 98C-127H segment does not have the characteristics of the classical zinc fingers.
大肠杆菌谷氨酰胺-tRNA合成酶片段的锌含量，以及仅在含锌谷氨酰胺-tRNA合成酶中CYC序列的保守性，表明98CYCX24-CRHSHEHHADDEPC138包含一些或全部参与结合其锌原子的残基（Liu, J., Lin, S.-X., Blochet, J.-E., Pézolet, M., and Lapointe, J. (1993) Biochemistry 32, 11390-11396）。扩展X射线吸收精细结构（EXAFS）表明，该锌原子具有一个四配位的非平面配位环境，有3个硫原子和1个氮原子，键长分别为2.37±0.02 Å和2.01±0.02 Å，推测分别属于3个半胱氨酸残基和1个组氨酸残基。将98C-138C片段中的每个组氨酸和半胱氨酸残基分别保守替换为谷氨酰胺（Q）和丝氨酸（S），产生变体H129Q、H131Q、H132Q和C138S（它们能维持谷氨酸-tRNA合成酶对温度敏感的大肠杆菌JP1449在42℃下的生长）以及C98S、C100S、C125S和H127Q（它们不能）。这些突变体中这种酶的量比野生型菌株中至少大1个数量级；然而，在变体C100S和C125S的提取物中未检测到谷氨酰胺-tRNA合成酶活性，而在C98S和H127Q的提取物中其比活性比过量产生野生型酶或变体H129Q、H131Q、H132Q和C138S的细胞中低约10倍。这些结果表明，大肠杆菌谷氨酰胺-tRNA合成酶中存在的锌原子由98和100位的2个进化保守半胱氨酸以及Cys125和His127结合。利用大肠杆菌谷氨酰胺-tRNA合成酶的已知结构对该酶N端一半进行分子建模，支持了这一结论，并表明98C-127H片段不具有经典锌指的特征。
