Preliminary X-ray crystallographic analysis of an engineered glutamyl-tRNA synthetase from<i>Escherichia coli</i>

Chongdar, Nipa; Dasgupta, Saumya; Datta, Aritreyee; Basu, Gautam

doi:10.1107/s2053230x14010723

Cited by 4 publications

(5 citation statements)

References 29 publications

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“…17 More recently, the structure of an engineered form of GluRS from E. coli that contains two mutations (K236E/E328A) to allow crystallization has been solved. 18 There is moderate amino acid sequence conservation of GluRS from P. aeruginosa when compared with the corresponding enzymes from Bacillus subtilis, E. coli, T. elongatus , or Tth , with the number of conserved residues ranging between 35 and 43%. However, when compared with human mitochondrial GluRS (hmGluRS), there is only 30% conservation of the amino acid residues, and this drops to >12% when compared to the N-terminal 676 amino acids (which contains the GluRS activity) of human cytosolic Glu-ProRS ( Table S1 ).…”

Section: Resultsmentioning

confidence: 99%

Identification of Chemical Compounds That Inhibit the Function of Glutamyl-tRNA Synthetase from Pseudomonas aeruginosa

Hu¹,

Guerrero²,

Keniry³

et al. 2015

SLAS Discovery

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Pseudomonas aeruginosa glutamyl-tRNA synthetase (GluRS) was overexpressed in Escherichia coli. Sequence analysis indicated that P. aeruginosa GluRS is a discriminating GluRS and, similar to other GluRS proteins, requires the presence of tRNAGlu to produce a glutamyl-AMP intermediate. Kinetic parameters for interaction with tRNA were determined and the kcat and KM were 0.8 s−1 and 0.68 µM, respectively, resulting in a kcat/KM of 1.18 s−1 µM−1. A robust aminoacylation-based scintillation proximity assay (SPA) assay was developed and 800 natural products and 890 synthetic compounds were screened for inhibitory activity against P. aeruginosa GluRS. Fourteen compounds with inhibitory activity were identified. IC50s were in the low micromolar range. The minimum inhibitory concentration (MIC) was determined for each of the compounds against a panel of pathogenic bacteria. Two compounds, BT_03F04 and BT_04B09, inhibited GluRS with IC50s of 21.9 and 24.9 µM, respectively, and both exhibited promising MICs against Gram-positive bacteria. Time-kill studies indicated that one compound was bactericidal and one was bacteriostatic against Gram-positive bacteria. BT_03F04 was found to be noncompetitive with both ATP and glutamic acid, and BT_04B09 was competitive with glutamic acid but noncompetitive with ATP. The compounds were not observed to be toxic to mammalian cells in MTT assays.

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Section: Resultsmentioning

confidence: 99%

Identification of Chemical Compounds That Inhibit the Function of Glutamyl-tRNA Synthetase from Pseudomonas aeruginosa

Hu¹,

Guerrero²,

Keniry³

et al. 2015

SLAS Discovery

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“…Among the six available structures pZBD s present in bacterial GluRS ( Table 1 ) only Bb -GluRS is zinc-bound, chelated by the CxCx 20–21 Yx 3 C motif (bold letters indicate co-ordinating residues). Ec -GluRS, for which only a preliminary crystallization report is available [ 15 ], also displays an identical ZB-motif CxCx 20 Yx 3 C (the histidine mentioned in the introduction section appears after the motif as: CxCx 20 Yx 3 CxH). The bound Zn 2+ in Ec- GluRS was shown to be functionally important [ 9 , 10 , 12 ].…”

Section: Resultsmentioning

confidence: 99%

“…A previously reported [ 15 ] Ec -GluRS encoding plasmid was used for the construction of pZBD -chimeras. Four sets of oligonucleotides were designed and were purchased from Integrated DNA Technologies.…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2015

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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.

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“…Τηε προτειν πυριφιςατιον ανδ ςρψσταλλιζατιον οφ αν ενγινεερεδΕςο -ΓλυΡΣ (Κ236Ε/Ε328Α) ωας πρε ιουσλψ ρεπορτεδ (27). Σινςε τηε ςορρεσπονδινγ ωιλδ τψπε ΓλυΡΣ φαιλεδ το ςρψσταλλιζε δεσπιτε μυλτιπλε αττεμπτς υνδερ δι ερσε ςονδιτιονς, ΓλυΡΣ στρυςτυρε ωας σολ εδ υσινγ τηε πρε ιουσλψ ςολλεςτεδ δατα (ρεσολυτιον υπ το 3.3 Å).…”

Section: Principal Component Analysisunclassified

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

Basu

Dasgupta

Dev

et al. 2023

Preprint

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

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

Cited by 4 publications

References 29 publications

Identification of Chemical Compounds That Inhibit the Function of Glutamyl-tRNA Synthetase from Pseudomonas aeruginosa

Identification of Chemical Compounds That Inhibit the Function of Glutamyl-tRNA Synthetase from Pseudomonas aeruginosa

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

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

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