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  • amiodarone hcl Bacterial GluRS comes in two flavors discrimi

    2021-11-26

    Bacterial GluRS comes in two flavors, discriminatory GluRS (D-GluRS) and non-discriminatory GluRS (ND-GluRS). While D-GluRS exclusively catalyses the transfer of Glu to tRNAGlu, the ND-GluRS can also glutamylate tRNAGln forming Glu-tRNAGln. The misacylated product is then transformed to Gln-tRNAGln by an enzyme known as glutamyl-tRNAGln amidotransferase [5], [6]. Identification of key residues (identity elements) in GluRS, GlnRS and the corresponding tRNAs that play a crucial role in aminoacylation showed that the participation of both domains is required for efficient aminoacylation of tRNAGlu by D-GluRS and tRNAGln by GlnRS [7], amiodarone hcl [8], [9], [10]. However, tRNA recognition pattern of these two enzymes to their cognate tRNAs are different. For GluRS–tRNAGlu interaction, the identity elements were mainly clustered in the catalytic domain of GluRS and on the acceptor arm and augmented D-helix of tRNAGlu[8]. For GlnRS–tRNAGln interaction, the identity elements were widely spread in both the domains of GlnRS and in tRNAGln[9]. In amiodarone hcl to the wide range of data available for cognate tRNA interaction [7], [8], [9], [10], information about non-cognate tRNA discrimination is scanty and is focused only on the role of the anticodon-binding domain [11], [12], [13]. Sekine et al. [11] showed that for Thermus thermophilus GluRS (TtGluRS), Arg358 (at position R) is responsible for tRNAGln discrimination, because an Arg358Gln mutation resulted in a GluRS that showed relaxed specificity towards the anticodons of tRNAGlu/Gln (34CUC36/34CUG36). Although this Arg residue is conserved in D-GluRS, several ND-GluRSs are also known to contain an Arg residue at this position. Based on limited sequence alignment it was suggested that an Arg at position R is allowed in ND-GluRS provided there is no Glu at another upstream position (Glu443 in TtGluRS) [12]. However, this rule is not universal (GluRS in Thermotogae petrophila, non-discriminatory due to the absence of GlnRS in the genome and the concomitant presence of GatCAB genes, contains an Arg-Glu pair at positions 358/443 corresponding to TtGluRS). That the Arg residue is not the sole player in tRNA discrimination is also evident from the work of Lee and Hendrickson [13]. A mutation at position R (Arg350Glu) in Helicobacter pylori GluRS1, a canonical D-GluRS, did not affect discrimination against tRNAGln and produced Glu-tRNAGlu like the wild type protein. On the other hand, introduction of an Arg residue at position R in H. pylori GluRS2 (Glu334Arg), a non-canonical GluRS that produces misacylated Glu-tRNAGln but does not produce Glu-tRNAGlu, did not nullify its discrimination against tRNAGlu but showed weak glutamylation of tRNAGln. Another single mutation (Gly417Thr) in H. pylori GluRS2 produced weak activity towards tRNAGlu while a double mutant (Glu334Arg, Gly417Thr) more robustly glutamylated tRNAGlu instead of tRNAGln, with undetectable production of Glu-tRNAGln for both cases. The experimental data show that tRNA discrimination by the anticodon-binding domain of GluRS is complex and operates in synergy among more than one residue positions. It is also likely that the catalytic domain of GluRS plays a role in tRNA discrimination, since tRNAGlu as well as tRNAGln contain identity elements not only in the anticodon loop but also in the augmented D-loop and the acceptor arm [7], [8], [9], [10]. The contribution of the D-GluRS catalytic domain in discrimination against tRNAGln is an open question that needs to be addressed. We have compared the properties of Escherichia coli GluRS (EcGluRS; D-GluRS) and its two domains, the N-terminal catalytic domain of GluRS (NGluRS) and the C-terminal anticodon-binding domain of GluRS (CGluRS), to probe the contributions of the two domains in terms of cognate (EctRNAGlu) and non-cognate (EctRNAGln) glutamylation. In addition, tRNAGln discrimination by a previously reported chimeric protein, chimera of NGluRS and the anticodon-binding domain of GlnRS (cGluGlnRS) [14], constructed from NGluRS and the anticodon-binding domain of EcGlnRS was also studied for comparison. Our results show that the N-terminal catalytic domain discriminates against tRNAGln demonstrating that in addition to the anticodon-binding domain, anti-determinants are also present in the catalytic domain of EcGluRS.