Seryl-tRNA synthetase from Escherichia coli: implication of its N-terminal domain in aminoacylation activity and specificity.
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.
Borel
F
F
European Molecular Biology Laboratory, Grenoble, France.
Vincent
C
C
Leberman
R
R
Härtlein
M
M
D000215:Acylation; D000255:Adenosine Triphosphate / Q000378:metabolism; D001665:Binding Sites; D004926:Escherichia coli / Q000201:enzymology*; D017353:Gene Deletion; D007700:Kinetics; D046911:Macromolecular Substances; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017433:Protein Structure, Secondary; D012362:RNA, Transfer, Ser / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D012714:Serine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
D046911:Macromolecular Substances; D012362:RNA, Transfer, Ser; D012694:Serine; D000255:Adenosine Triphosphate; D012714:Serine-tRNA Ligase