Open AccessAn Example of Non‐Conservation of Oligomeric Structure in Prokaryotic Aminoacyl‐tRNA Synthetases
Author(s) -
Mazauric MarieHélène,
Reinbolt Joseph,
Lorber Bernard,
Ebel Christine,
Keith Gérard,
Giegé Richard,
Kern Daniel
Publication year - 1996
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1111/j.1432-1033.1996.00814.x
Subject(s) - thermus thermophilus , transfer rna , biology , biochemistry , aminoacyl trna synthetase , escherichia coli , aminoacylation , thermophile , amino acyl trna synthetases , enzyme , thermostability , rna , gene
Glycyl‐tRNA synthetase (Gly‐tRNA synthetase) from Thermus thermophilus was purified to homogeneity and with high yield using a five‐step purification procedure in amounts sufficient to solve its crystallographic structure [Logan, D. T., Mazauric, M.‐H., Kern, D. & Moras, D. (1995) EMBO J. 14 , 4156–4167]. Molecular‐mass determinations of the native and denatured protein indicate an oligomeric structure of the α 2 type consistent with that found for eukaryotic Gly‐tRNA synthetases (yeast and Bombyx mori ), but different from that of Gly‐tRNA synthetases from mesophilic prokaryotes ( Escherichia coli and Bacillus brevis ) which are α 2 β 2 tetramers. N‐terminal sequencing of the polypeptide chain reveals significant identity, reaching 50% with those of the eukaryotic enzymes ( B. mori, Homo sapiens , yeast and Caenorhabditis elegans ) but no significant identity was found with both α and β chains of the prokaryotic enzymes ( E. coli, Haemophilus influenzue and Coxiella burnetii ) albeit the enzyme is deprived of the N‐terminal extension characterizing eukaryotic synthetases. Thus, the thermophilic Gly‐tRNA synthetase combines strong structural homologies of eukaryotic Gly‐tRNA synthetases with a feature of prokaryotic synthetases. Heat‐stability measurements show that this synthetase keeps its ATP‐PP i exchange and aminoacylation activities up to 70°C. Glycyladenylate strongly protects the enzyme against thermal inactivation at higher temperatures. Unexpectedly, tRNA Gly does not induce protection. Cross‐aminoacylations reveal that the thermophilic Gly‐tRNA synthetase charges heterologous E. coli tRNA Gly(GCC) and tRNA Gly(CCC) and yeast tRNA Gly(GCC) as efficiently as T. thermophilus tRNA Gly . All these aminoacylation reactions are characterized by similar activation energies as deduced from Arrhenius plots. Therefore, contrary to the E. coli and H. sapiens Gly‐tRNA synthetases, the prokaryotic thermophilic enzyme does not possess a strict species specificity. The results are discussed in the context of the three‐dimensional structure of the synthetase and in the view of the particular evolution of the glycinylation systems.