Dispensability of zinc and the putative zinc-binding domain in bacterial glutamyl-tRNA synthetase

Abstract

The putative zinc-binding domain (pZBD) in Escherichia coli glutamyl-tRNA synthetase (GluRS) is known to correctly position the tRNA acceptor arm and modulate the amino acid-binding site. However, its functional role in other bacterial species is not clear since many bacterial GluRSs lack a zinc-binding motif in the pZBD. From experimental studies on pZBD-swapped E. coli GluRS, with Thermosynechoccus elongatus GluRS, Burkholderia thailandensis GluRS and E. coli glutamyl-queuosine-tRNAAsp synthetase (Glu-Q-RS), we show that E. coli GluRS, containing the zinc-free pZBD of B. thailandensis, is as functional as the zinc-bound wild-type E. coli GluRS, whereas the other constructs, all zinc-bound, show impaired function. A pZBD-tinkered version of E. coli GluRS that still retained Zn-binding capacity, also showed reduced activity. This suggests that zinc is not essential for the pZBD to be functional. From extensive structural and sequence analyses from whole genome database of bacterial GluRS, we further show that in addition to many bacterial GluRS lacking a zinc-binding motif, the pZBD is actually deleted in some bacteria, all containing either glutaminyl-tRNA synthetase (GlnRS) or a second copy of GluRS (GluRS2). Correlation between the absence of pZBD and the occurrence of glutamine amidotransferase CAB (GatCAB) in the genome suggests that the primordial role of the pZBD was to facilitate transamidation of misacylated Glu-tRNAGln via interaction with GatCAB, whereas its role in tRNAGlu interaction may be a consequence of the presence of pZBD.

Signatures of tRNA Glx -specificity in bacterial glutamyl-tRNA synthetases

“…Residue His127, which was proposed to be the fourth coordinating ligand of Zn 2+ points away from it. This observation supports our previous report (20), where we had predicted the coordinating ligands of zinc in Eco -GluRS to be Cys98, Cys100, Cys125 and Tyr121, based on its sequence similarity to GluRS from Borrelia burgdorferri (Bbu -GluRS; PDB ID: 4gri), the only other bacterial GluRS structure that contains a Zn 2+ (22). It is worth mentioning here that the coordination environment of Zn 2+ in Eco -GluRS is similar to that of YadB gene product of E. coli (23,24),Eco -Glu-Q-RS (pdb ID: 1nzj), the N-terminal only paralogue of GluRS.…”

“…Interestingly, this is not true in case of non-proteobacterium T. thermophilus , which, despite possessing a D-GluRS, displays augmented D-helix in both tRNA Glu and in tRNA Gln . A zinc ion present in the catalytic domain of Eco -GluRS was shown to play a critical role glutamylation reaction (19), although many bacterial GluRSs do not contain a bound Zn 2+ , including Tth -GluRS, implying the irregular occurrence of the zinc atom (20). In another study, when an arginine residue (R266) in the tRNA-binding interface of Eco -GluRS was mutated to leucine, glutamylation efficiency of the protein was drastically reduced (more than 2500 fold) (16).…”

The zinc-binding domain of mammalian prolyl-tRNA synthetase is indispensable for catalytic activity and organism viability

“…Zn 2+ content of recombinant human wild-type and ΔZ mutant ProRS was determined by Zn 2+ release by methyl methanethiosulfonate and spectroscopic detection with 4-(2-pyridylazo)resorcinol ( Chongdar et al., 2015 ; Hunt et al., 1985 ). Unlike substantial Zn 2+ content of wild-type enzyme ( Figure 4 F, left), Zn 2+ in the ΔZ mutant was virtually undetectable, consistent with the absence of two coordinating cysteine ligands ( Figure 4 F, right).…”

Zinc binding proteome of a phytopathogen Xanthomonas translucens pv. undulosa

“…Five of having tRNA synthetase II, one has ribosomal L2 and one has ribosomal S12/S23 domain. It was previously found that Zn ion helps in structural stability of these identified domains, which are involved in protein biosynthesis and also act as targets for many biocontrol agents [109][110][111]144]. A total of four proteins were found in the class of response to oxidative stress.…”