Leucyl-tRNA synthetase consisting of two subunits from hyperthermophilic bacteria Aquifex aeolicus.
In a hyperthermophilic bacterium, Aquifex aeolicus, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (alpha and beta), different from the canonical LeuRS, which has a single polypeptide chain. By PCR, using genome DNA of A. aeolicus as a template, genes encoding the alpha and beta subunits were amplified and cloned in Escherichia coli. The alpha subunit could not be expressed stably in vivo, whereas the beta subunit was overproduced and purified by a simple procedure. The beta subunit was inactive in catalysis but was able to bind tRNA(Leu). Interestingly, the heterodimer alphabeta-LeuRS could be overproduced in E. coli cells containing both genes and was purified to 95% homogeneity as a hybrid dimer. The kinetics of A. aeolicus LeuRS in pre-steady and steady states and cross-recognition of LeuRS and tRNA(Leu) from A. aeolicus and E. coli were studied. Magnesium concentration, pH value, and temperature aminoacylation optima were determined to be 12 mm, 7.8, and 70 degrees C, respectively. Under optimal conditions, A. aeolicus alphabeta-LeuRS is stable up to 65 degrees C.
State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, China.
Min-Gang
MG
Xu
Jian-Feng
JF
Chen
Franck
F
Martin
Ming-Wei
MW
Zhao
Gilbert
G
Eriani
En-Duo
ED
Wang
D001419:Bacteria / Q000201:enzymology*; D001665:Binding Sites; D002942:Circular Dichroism; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli / Q000201:enzymology; D007700:Kinetics; D007935:Leucine-tRNA Ligase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification; D021122:Protein Subunits; D012356:RNA, Transfer, Leu
D021122:Protein Subunits; D012356:RNA, Transfer, Leu; D007935:Leucine-tRNA Ligase