Split leucine-specific domain of leucyl-tRNA synthetase from the hyperthermophilic bacterium Aquifex aeolicus.
Leucyl-tRNA synthetase (LeuRS) from Aquifex aeolicus is the only known heterodimer synthetase. It is named LeuRS alphabeta;, and its alpha and beta subunits contain 634 and 289 residues, respectively. Like Thermus thermophilus LeuRS, LeuRS alphabeta has a large extra domain, the leucine-specific domain, inserted into the catalytic domain. The subunit split site is exactly in the middle of the leucine-specific domain and may have a unique function. Here, a series of mutants of LeuRS alphabeta consisting of either mutated alpha subunits and wild-type beta subunits or wild-type alpha subunits and mutated beta subunits were constructed and purified. ATP-PPi exchange and aminoacylation activities and the ability of the mutants to charge minihelix(Leu) were assayed. Interaction of the mutants with the tRNA was assessed by gel shift. Two peptides of eight and nine amino acid residues in the domain located in the alpha subunit were found to be essential for the enzyme's activity. We also showed that the domain in LeuRS alphabeta plays an important role in minihelix(Leu) recognition. Additionally, the domain was found to have little impact on the assembly of the heterodimer, to play a role in the thermal stability of the whole enzyme, and to interact with the cognate tRNA in the predicted manner.
Ma
Jing-Jing
JJ
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Graduate School of the Chinese Academy of Sciences, 320 Yue Yang Road, Shanghai 200031, People's Republic of China.
Zhao
Ming-Wei
MW
Wang
En-Duo
ED
D019943:Amino Acid Substitution; D001419:Bacteria / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D007930:Leucine / Q000032:analysis*; D007935:Leucine-tRNA Ligase / Q000737:chemistry* / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010957:Plasmids; D021122:Protein Subunits; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics
D001426:Bacterial Proteins; D021122:Protein Subunits; D011994:Recombinant Proteins; D007935:Leucine-tRNA Ligase; D007930:Leucine