Igor Živković1, Karla Knežević1, Ita Gruić Sovulj1
1Department of Chemistry, Faculty of Science, University of Zagreb, Zagreb, Croatia
Aminoacyl-tRNA synthetases (AARS) are enzymes responsible for attaching amino acids to their cognate tRNA molecules, thus providing the required substrates for protein synthesis. Aminoacylation occurs in two steps: amino acid is firstly activated with ATP to form AA-AMP, followed by transfer of the amino acyl moiety to the tRNA. AARS are divided into two evolutionary distinct classes, I and II. Class I AARS feature a highly conserved signature sequence His-Ile-Gly-His (HIGH motif) responsible for the binding of ATP and stabilization of the transition state for the amino acid activation. Recently, we have observed that isoleucyl-tRNA synthetase (IleRS), class I AARS, may possess variations of the HIGH motif (HIGH to GIHH exchange) related to the resistance to natural antibiotic mupirocin, which binds to the IleRS synthetic active site. While typically highly conserved throughout all class I AARS, the HIGH motif can undergo natural variations in some rare cases, leading to the emergence of novel and interesting features. Following this idea, we examined sequences of many class I AARS and found that besides IleRS only arginyl-tRNA synthetase (ArgRS) may also comprise variations in the HIGH motif. Two interesting variations have been found: VLVQ in Symbiobacterium thermophilum ArgRS (StArgRS) and RVDH in Herpetosiphon aurantiacus ArgRS (HaArgRS). The observed motif variations are more extreme than those previously studied in IleRS. Hence, unravelling whether the corresponding ArgRS enzymes acquired some new and unique features by the atypical motifs is highly relevant. To answer these questions, we cloned and expressed the aforementioned ArgRSs in E. coli and purified them for kinetic and structural characterisation. Preliminary kinetical analysis shows that all tested ArgRSs are active in the aminoacylation reaction. However, we found that ArgRS are generally prone to the formation of soluble aggregates that preclude efficient purification of the enzymes in large quantities for crystallization. By using different E. coli expression strains, coexpression with chaperones, varying the conditions of the expression, cell lysis and purification techniques and conditions we were able to increase the monomer fractions to a suitable amount for crystallization.
Keywords: arginyl-tRNA synthetase; catalytic motif; herpetosiphon; symbiobacterium thermophilum