Structural and functional consequences of mutating a proteobacteria‐specific surface residue in the catalytic domain of <i>Escherichia coli</i> GluRS

Dasgupta, Saumya; Manna, Debasis; Basu, Gautam

doi:10.1016/j.febslet.2012.05.006

Cited by 5 publications

(10 citation statements)

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“…These GluRS sequences appear in the non-proteobacterial cluster, as sister clades of chlamydiae, fusobacteria and deinococcous-thermus. Unlike the canonical proteobacterial GluRS (the grey shaded region of Figure 2), GluRS belonging to the γ*-/α*-group seem to have appeared through some alternate evolutionary route, probably via HGT, as has been noted earlier [22]. Interestingly, in gatB phylogeny (Figure 3) the gatB sequences of the γ*-/α*-group are not outliers, indicating that only GluRS and not gatB appeared by HGT in these bacteria.…”

Section: Resultsmentioning

confidence: 57%

“…Such phylum-specific trends have been observed experimentally for GluRS-tRNA Gln interaction – a D-GluRS-specific residue (Arg358) in Thermus thermophilus GluRS led to a relaxed tRNA Gln -discrimination [45] but when the same residue was mutated in H. pylori (GluRS1), no such effect was observed [46]. Similarly, for GluRS-tRNA Glu interaction, it was found that a proteobacteria-specific Arg residue (Arg 266 in E. coli GluRS) was absolutely essential for glutamylation efficiency of GluRS but the Arg is replaced by mostly Leu in non-proteobacterial GluRS [22]. …”

Section: Resultsmentioning

confidence: 99%

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Evolutionary insights about bacterial GlxRS from whole genome analyses: is GluRS2 a chimera?

Dasgupta

Basu

2014

BMC Evol Biol

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BackgroundEvolutionary histories of glutamyl-tRNA synthetase (GluRS) and glutaminyl-tRNA synthetase (GlnRS) in bacteria are convoluted. After the divergence of eubacteria and eukarya, bacterial GluRS glutamylated both tRNAGln and tRNAGlu until GlnRS appeared by horizontal gene transfer (HGT) from eukaryotes or a duplicate copy of GluRS (GluRS2) that only glutamylates tRNAGln appeared. The current understanding is based on limited sequence data and not always compatible with available experimental results. In particular, the origin of GluRS2 is poorly understood.ResultsA large database of bacterial GluRS, GlnRS, tRNAGln and the trimeric aminoacyl-tRNA-dependent amidotransferase (gatCAB), constructed from whole genomes by functionally annotating and classifying these enzymes according to their mutual presence and absence in the genome, was analyzed. Phylogenetic analyses showed that the catalytic and the anticodon-binding domains of functional GluRS2 (as in Helicobacter pylori) were independently acquired from evolutionarily distant hosts by HGT. Non-functional GluRS2 (as in Thermotoga maritima), on the other hand, was found to contain an anticodon-binding domain appended to a gene-duplicated catalytic domain. Several genomes were found to possess both GluRS2 and GlnRS, even though they share the common function of aminoacylating tRNAGln. GlnRS was widely distributed among bacterial phyla and although phylogenetic analyses confirmed the origin of most bacterial GlnRS to be through a single HGT from eukarya, many GlnRS sequences also appeared with evolutionarily distant phyla in phylogenetic tree. A GlnRS pseudogene could be identified in Sorangium cellulosum.ConclusionsOur analysis broadens the current understanding of bacterial GlxRS evolution and highlights the idiosyncratic evolution of GluRS2. Specifically we show that: i) GluRS2 is a chimera of mismatching catalytic and anticodon-binding domains, ii) the appearance of GlnRS and GluRS2 in a single bacterial genome indicating that the evolutionary histories of the two enzymes are distinct, iii) GlnRS is more widespread in bacteria than is believed, iv) bacterial GlnRS appeared both by HGT from eukarya and intra-bacterial HGT, v) presence of GlnRS pseudogene shows that many bacteria could not retain the newly acquired eukaryal GlnRS. The functional annotation of GluRS, without recourse to experiments, performed in this work, demonstrates the inherent and unique advantages of using whole genome over isolated sequence databases.

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Section: Resultsmentioning

confidence: 57%

Section: Resultsmentioning

confidence: 99%

Evolutionary insights about bacterial GlxRS from whole genome analyses: is GluRS2 a chimera?

Dasgupta

Basu

2014

BMC Evol Biol

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“…The diverse tRNA Glx specificities of bacterial GluRS call for structure-function correlation studies that will yield a clear understanding of the mechanisms that control the GluRS-tRNA Glx interaction in bacteria. Because the precise nature of the GluRS-tRNA Glx interaction in bacteria is phylum-specific (Dasgupta et al, 2012;Dasgupta & Basu, 2014), biochemical work performed on the GluRS-tRNA Glx interaction in a bacterium from one phylum may not be compatible with the GluRS structure from a bacterium belonging to a distant phylum.…”

Section: Introductionmentioning

confidence: 99%

“…Escherichia coli is a model bacterium, some strains of which are pathogenic. Most biochemical studies have been performed on E. coli GluRS (Eco-GluRS), both in our laboratory (Dasgupta et al, 2009(Dasgupta et al, , 2012Saha et al, 2009Saha et al, , 2012 and in those of others (Sekine et al, 1996;Banerjee et al, 2004;Dubois et al, 2009). Recent reports have demonstrated how Eco-GluRS plays a role in pathogenesis by inducing multidrug tolerance upon binding to a eukaryote-like serine-threonine kinase (HipA; Germain et al, 2013;Kaspy et al, 2013).…”

Section: Introductionmentioning

confidence: 99%

“…The homology model of Eco-GluRS is not precise enough to reveal the subtleties of GluRS-tRNA Glu interaction in E. coli owing to the absence of some specific features of Eco-GluRS in the template structures. For example, the proteobacterial-specific residue Arg266, which is present in a tRNA-interacting loop of Eco-GluRS (Dasgupta et al, 2012), is absent in all known bacterial GluRS structures except for that of B. thailandensis GluRS (Bth-GluRS; see Fig. 1b).…”

Section: Introductionmentioning

confidence: 99%

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Preliminary X-ray crystallographic analysis of an engineered glutamyl-tRNA synthetase fromEscherichia coli

et al. 2014

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The nature of interaction between glutamyl-tRNA synthetase (GluRS) and its tRNA substrate is unique in bacteria in that many bacterial GluRS are capable of recognizing two tRNA substrates: tRNA Glu and tRNA Gln . To properly understand this distinctive GluRS-tRNA interaction it is important to pursue detailed structure-function studies; however, because of the fact that tRNAGluRS interaction in bacteria is also associated with phylum-specific idiosyncrasies, the structure-function correlation studies must also be phylumspecific. GluRS from Thermus thermophilus and Escherichia coli, which belong to evolutionarily distant phyla, are the biochemically best characterized. Of these, only the structure of T. thermophilus GluRS is available. To fully unravel the subtleties of tRNA Glu -GluRS interaction in E. coli, a model bacterium that can also be pathogenic, determination of the E. coli GluRS structure is essential. However, previous attempts have failed to crystallize E. coli GluRS. By mapping crystal contacts of a homologous GluRS onto the E. coli GluRS sequence, two surface residues were identified that might have been hindering crystallization attempts. Accordingly, these two residues were mutated and crystallization of the double mutant was attempted. Here, the design, expression, purification and crystallization of an engineered E. coli GluRS in which two surface residues were mutated to optimize crystal contacts are reported.

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Signatures of tRNA^Glx‐specificity in proteobacterial glutamyl‐tRNA synthetases

Dasgupta,

Dev,

Chongdar

et al. 2023

Proteins

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The canonical function of glutamyl‐tRNA synthetase (GluRS) is to glutamylate tRNAGlu. Yet not all bacterial GluRSs glutamylate tRNAGlu; many glutamylate both tRNAGlu and tRNAGln, while some glutamylate only tRNAGln and not the cognate substrate tRNAGlu. Understanding the basis of the unique specificity of tRNAGlx is important. Mutational studies have hinted at hotspot residues, both on tRNAGlx and GluRS, which play crucial roles in tRNAGlx‐specificity. However, its underlying structural basis remains unexplored. The majority of biochemical studies related to tRNAGlx‐specificity have been performed on GluRS from Escherichia coli and other proteobacterial species. However, since the early crystal structures of GluRS and tRNAGlu‐bound GluRS were from non‐proteobacterial species (Thermus thermophilus), proteobacterial biochemical data have often been interpreted in the context of non‐proteobacterial GluRS structures. Marked differences between proteobacterial and non‐proteobacterial GluRSs have been demonstrated; therefore, it is important to understand tRNAGlx‐specificity vis‐a‐vis proteobacterial GluRS structures. To this end, we solved the crystal structure of a double mutant GluRS from E. coli. Using the solved structure and several other currently available proteo‐ and non‐proteobacterial GluRS crystal structures, we probed the structural basis of the tRNAGlx‐specificity of bacterial GluRSs. Specifically, our analyses suggest a unique role played by the tRNAGlx D‐helix contacting loop of GluRS in the modulation of tRNAGln‐specificity. While earlier studies have identified functional hotspots on tRNAGlx that control the tRNAGlx‐specificity of GluRS, this is the first report of complementary signatures of tRNAGlx‐specificity in GluRS.

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Structural and functional consequences of mutating a proteobacteria‐specific surface residue in the catalytic domain of Escherichia coli GluRS

Cited by 5 publications

References 36 publications

Evolutionary insights about bacterial GlxRS from whole genome analyses: is GluRS2 a chimera?

Evolutionary insights about bacterial GlxRS from whole genome analyses: is GluRS2 a chimera?

Preliminary X-ray crystallographic analysis of an engineered glutamyl-tRNA synthetase fromEscherichia coli

Signatures of tRNA^Glx‐specificity in proteobacterial glutamyl‐tRNA synthetases

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Structural and functional consequences of mutating a proteobacteria‐specific surface residue in the catalytic domain of Escherichia coli GluRS

Cited by 5 publications

References 36 publications

Evolutionary insights about bacterial GlxRS from whole genome analyses: is GluRS2 a chimera?

Evolutionary insights about bacterial GlxRS from whole genome analyses: is GluRS2 a chimera?

Preliminary X-ray crystallographic analysis of an engineered glutamyl-tRNA synthetase fromEscherichia coli

Signatures of tRNAGlx‐specificity in proteobacterial glutamyl‐tRNA synthetases

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Signatures of tRNA^Glx‐specificity in proteobacterial glutamyl‐tRNA synthetases