Leucyl-tRNA synthetase consisting of two subunits from hyperthermophilic bacteria Aquifex aeolicus

Abstract

In a hyperthermophilic bacterium, Aquifex aeolicus, leucyl-tRNA synthetase (LeuRS) consists of two non-identical polypeptide subunits (α and β), 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 α and β subunits were amplified and cloned in Escherichia coli. The α subunit could not be expressed stably in vivo, whereas the β subunit was overproduced and purified by a simple procedure. The β subunit was inactive in catalysis but was able to bind tRNALeu. Interestingly, the heterodimer αβ-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 presteady and steady states and cross-recognition of LeuRS and tRNALeu 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 °C, respectively. Under optimal conditions, A. aeolicus αβ-LeuRS is stable up to 65 °C.