In-vivo protein nitration facilitates Vibrio cholerae cell survival under anaerobic, nutrient deprived conditions

Patra, Sourav Kumar; Sinha, Nilanjan; Molla, Firoz; Sengupta, Arunava; Chakraborty, Subrata; Roy, Souvik; Ghosh, Sudakshina

doi:10.1016/j.abb.2022.109358

Cited by 3 publications

(4 citation statements)

References 47 publications

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“…Finally, this technique may also be useful to reveal alterations of activity (if any) due to posttranslational modifications of aaRS proteins [23,25]. These post translational alterations might be related to oxidative [28] or nitrosative stress [29] or other stable biochemical modifications that occur in-vivo or in-vitro provided these alternations are not affected or reversed during zymography procedure of prestaining steps.…”

Section: Discussionmentioning

confidence: 99%

“…For semi-quantitative or qualitative analysis, a chromatic band intensity can be measured using ImageJ software [25] to determine relative activities of aaRS molecules in different samples.…”

Section: Methodsmentioning

confidence: 99%

“…Bacterial Cells were lysed, by sonication with 8 pulses of 10 second 70% amplitude sonic vibrations keeping 20 seconds of pause time, ensuring the tube remained in ice during sonication. Lysed cells were then centrifuged at 12000 rpm for 10 min at 4 °C and supernatants were collected, aliquoted in tubes, and stored at -80°C [20,[23][24][25].…”

Section: Cell Lysis Buffer Preparation and Cell Lysismentioning

confidence: 99%

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A Zymography technique to study amino acid activation by aminoacyl tRNA synthetases (aaRS): A broad spectrum, high-throughput tool to screen activities of aaRS and their “Urzyme” variants

Patra

Carter

2023

Preprint

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Amino acyl tRNA synthetases or aaRSs play a key role in assuring the precision of protein translation. They are highly specific for their cognate amino acid and cognate tRNA substrates during protein synthesis, utilizing ATP to ensure that proper assignments are made between amino acid and anticodon. Specific aaRS for each amino acid are present in all cells. We describe a new zymography technique to qualitatively visualize and semi-quantitatively determine the amino acid activation capacity of each type of aaRS molecule by indirect colorimetric detection of released pyrophosphates during the formation of aminoacyl-AMP. Protein samples containing aaRS are subjected to Native PAGE, followed by incubation in buffer containing cognate amino acid and ATP for sufficient time to generate pyrophosphates (PPi) which are then converted to inorganic phosphates by pyrophosphatase treatment. Finally, the generated and localized phosphates around the aaRS protein inside the gel can be visualized after staining by ammonium molybdate and malachite green solution. This technique has been validated by inspecting the substrate specificities of specific aaRSs. This zymography technique is sufficiently sensitive to detect and authenticate activities of much (i.e., ~10-5-fold) less active aaRS Urzymes, to study alteration of activities of aaRS by various intrinsic or extrinsic factors and to screen aaRS-specific antimicrobial drugs.

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Section: Discussionmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Cell Lysis Buffer Preparation and Cell Lysismentioning

confidence: 99%

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A Zymography technique to study amino acid activation by aminoacyl tRNA synthetases (aaRS): A broad spectrum, high-throughput tool to screen activities of aaRS and their “Urzyme” variants

Patra

Carter

2023

Preprint

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“…The following parameters were used to identify tryptic peptides: 10 ppm precursor ion mass tolerance; 0.02 Da product ion mass tolerance; up to 2 missed trypsin cleavage sites; (C) carbamidomethylation was set as a fixed modification; (M) oxidation was set as a variable modification. Peptide false discovery rates (FDR) were calculated by the Percolator node using a decoy database search and peptides were filtered using a 1% FDR cutoff 45 .…”

Section: Label Free Proteomics; Lc-esi-ms/ms Analysismentioning

confidence: 99%

Genomic database furnishes a spontaneous example of a functional Class II glycyl-tRNA synthetase urzyme

Patra,

Douglas,

Wills

et al. 2024

Preprint

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The chief barrier to studies of how genetic coding emerged is the lack of experimental models for ancestral aminoacyl-tRNA synthetases (AARS). We hypothesized that conserved core catalytic sites could represent such ancestors. That hypothesis enabled engineering functional urzymes from TrpRS, LeuRS, and HisRS. We describe here a fourth urzyme, GlyCA, detected in an open reading frame from the genomic record of the arctic fox, Vulpes lagopus. GlyCA is homologous to a bacterial heterotetrameric Class II GlyRS-B. Alphafold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to the HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment. Enzymatic characterization revealed a robust single-turnover burst size and a catalytic rate for ATP consumption well in excess of that previously published for HisCA1. Time-dependent aminoacylation of tRNAGly proceeds at a rate consistent with that observed for amino acid activation. In fact, GlyCA is actually 35 times more active in glycine activation by ATP than the full-length GlyRS-B α-subunit dimer. ATP-dependent activation of the 20 canonical amino acids favors Class II amino acids that complement those favored by HisCA and LeuAC. These properties reinforce the notion that urzymes represent the requisite ancestral catalytic activities to implement a reduced genetic coding alphabet.

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A genomic database furnishes minimal functional glycyl-tRNA synthetases homologous to other, designed class II urzymes

Patra,

Douglas,

Wills

et al. 2024

Nucleic Acids Research

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The hypothesis that conserved core catalytic sites could represent ancestral aminoacyl-tRNA synthetases (AARS) drove the design of functional TrpRS, LeuRS, and HisRS ‘urzymes’. We describe here new urzymes detected in the genomic record of the arctic fox, Vulpes lagopus. They are homologous to the α-subunit of bacterial heterotetrameric Class II glycyl-tRNA synthetase (GlyRS-B) enzymes. AlphaFold2 predicted that the N-terminal 81 amino acids would adopt a 3D structure nearly identical to our designed HisRS urzyme (HisCA1). We expressed and purified that N-terminal segment and the spliced open reading frame GlyCA1–2. Both exhibit robust single-turnover burst sizes and ATP consumption rates higher than those previously published for HisCA urzymes and comparable to those for LeuAC and TrpAC. GlyCA is more than twice as active in glycine activation by adenosine triphosphate as the full-length GlyRS-B α2 dimer. Michaelis–Menten rate constants for all three substrates reveal significant coupling between Exon2 and both substrates. GlyCA activation favors Class II amino acids that complement those favored by HisCA and LeuAC. Structural features help explain these results. These minimalist GlyRS catalysts are thus homologous to previously described urzymes. Their properties reinforce the notion that urzymes may have the requisite catalytic activities to implement a reduced, ancestral genetic coding alphabet.

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In-vivo protein nitration facilitates Vibrio cholerae cell survival under anaerobic, nutrient deprived conditions

Cited by 3 publications

References 47 publications

A Zymography technique to study amino acid activation by aminoacyl tRNA synthetases (aaRS): A broad spectrum, high-throughput tool to screen activities of aaRS and their “Urzyme” variants

A Zymography technique to study amino acid activation by aminoacyl tRNA synthetases (aaRS): A broad spectrum, high-throughput tool to screen activities of aaRS and their “Urzyme” variants

Genomic database furnishes a spontaneous example of a functional Class II glycyl-tRNA synthetase urzyme

A genomic database furnishes minimal functional glycyl-tRNA synthetases homologous to other, designed class II urzymes

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