Borel F, Vincent C, Leberman R, Härtlein M
European Molecular Biology Laboratory, Grenoble, France.
Nucleic Acids Res. 1994 Aug 11;22(15):2963-9. doi: 10.1093/nar/22.15.2963.
Escherichia coli seryl-tRNA synthetase (SerRS) a dimeric class II aminoacyl-tRNA synthetase with two structural domains charges specifically the five iso-acceptor tRNA(ser) as well as the tRNA(sec) (selC product) of E. coli. The N-terminal domain is a 60 A long arm-like coiled coil structure built of 2 long antiparallel a-h helices, whereas the C-terminal domain is a alpha-beta structure. A deletion of the N-terminal arm of the enzyme does not affect the amino acid activation step of the reaction, but reduces dramatically amino-acylation activity. The Kcat/Km value for the mutant enzyme is reduced by more than 4 orders of magnitude, with a nearly 30 fold increased Km value for tRNA(ser). An only slightly truncated mutant form (16 amino acids of the tip of the arm replaced by a glycine) has an intermediate aminoacylation activity. Both mutant synthetases have lost their specificity for tRNA(ser) and charge also non-cognate type 1 tRNA(s). Our results support the hypothesis that class II synthetases have evolved from an ancestral catalytic core enzyme by adding non-catalytic N-terminal or C-terminal tRNA binding (specificity) domains which act as determinants for cognate and anti-determinants for non-cognate tRNAs.
大肠杆菌丝氨酰 - tRNA合成酶（SerRS）是一种二聚体II类氨酰 - tRNA合成酶，具有两个结构域，它能特异性地将大肠杆菌的五种同工受体tRNA(ser)以及tRNA(sec)（selC产物）氨酰化。N端结构域是一个由2条长的反平行α - 螺旋构成的60 Å长的臂状卷曲螺旋结构，而C端结构域是一个α - β结构。该酶N端臂的缺失不影响反应的氨基酸活化步骤，但会显著降低氨酰化活性。突变酶的Kcat/Km值降低了4个多数量级，tRNA(ser)的Km值增加了近30倍。一种仅轻微截短的突变形式（臂末端的16个氨基酸被甘氨酸取代）具有中等氨酰化活性。两种突变合成酶都失去了对tRNA(ser)的特异性，并且也能将非同源的1型tRNA(s)氨酰化。我们的结果支持这样一种假说，即II类合成酶是通过添加非催化性的N端或C端tRNA结合（特异性）结构域从祖先催化核心酶进化而来的，这些结构域充当同源tRNA的决定因素和非同源tRNA的反决定因素。
