Covalent methionylation of <i>escherichia coli</i> methionyl‐tRNA synthethase: Identification of the labeled amino acid residues by matrix‐assisted laser desorption‐ionization mass spectrometry

Gillet, Sylvie; Hountondji, Codjo; Schmitter, Jean‐Marie; Blanquet, Sylvain

doi:10.1002/pro.5560061116

Cited by 18 publications

(13 citation statements)

References 46 publications

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“…The consistency among the different assays suggests that despite the selfmethionylation by the enzyme, the kinetics of aminoacylation in the presence of tRNA is unaffected. This is consistent with the notion that the self-methionylation reaction occurs at a much slower rate relative to aminoacylation (Gillet et al 1997), so that the self-methionylation was not detected in the time scale of one turnover of aminoacylation.…”

supporting

confidence: 90%

“…A set of mixing controls established that the observed reaction rates were independent of the premixing of the reactants. Additionally, because EcMetRS has an ability to self-methionylate to lysine residues near the active site (Gillet et al 1997), as evidenced in the self-labeling by covalent incorporation of 35 S-Met to the enzyme in both the presence and absence of tRNA Met (Supplemental Fig. S1A,B), aminoacylation was also monitored by two different methods.…”

Section: Kinetic Analysis Of Ecmetrsmentioning

confidence: 99%

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Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Liu¹,

Sanders²,

Pascal³

et al. 2011

RNA

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Class I aminoacyl-tRNA synthetases (aaRSs) use a Rossmann-fold domain to catalyze the synthesis of aminoacyl-tRNAs required for decoding genetic information. While the Rossmann-fold domain is conserved in evolution, the acceptor stem near the aminoacylation site varies among tRNA substrates, raising the question of how the conserved protein fold adapts to RNA sequence variations. Of interest is the existence of an unpaired C-A mismatch at the 1-72 position unique to bacterial initiator tRNA fMet and absent from elongator tRNAs. Here we show that the class I methionyl-tRNA synthetase (MetRS) of Escherichia coli and its close structural homolog cysteinyl-tRNA synthetase (CysRS) display distinct patterns of recognition of the 1-72 base pair. While the structural homology of the two enzymes in the Rossmann-fold domain is manifested in a common burst feature of aminoacylation kinetics, CysRS discriminates against unpaired 1-72, whereas MetRS lacks such discrimination. A structurebased alignment of the Rossmann fold identifies the insertion of an a-helical motif, specific to CysRS but absent from MetRS, which docks on 1-72 and may discriminate against mismatches. Indeed, substitutions of the CysRS helical motif abolish the discrimination against unpaired 1-72. Additional structural alignments reveal that with the exception of MetRS, class I tRNA synthetases contain a structural motif that docks on 1-72. This work demonstrates that by flexible insertion of a structural motif to dock on 1-72, the catalytic domain of class I tRNA synthetases can acquire structural plasticity to adapt to changes at the end of the tRNA acceptor stem.

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supporting

confidence: 90%

Section: Kinetic Analysis Of Ecmetrsmentioning

confidence: 99%

Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Liu¹,

Sanders²,

Pascal³

et al. 2011

RNA

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“…The low levels of self biotinylation and promiscuous protein biotinylation by wild type BirA observed in vivo seems likely to be a consequence of over expression of the enzyme resulting in an intracellular BirA concentration that greatly exceeds the supply of apo-BCCP, the normal biotin acceptor. In vitro methionyl-tRNA synthetase self modification is suppressed by addition of the tRNA acceptor (Gillet et al 1997) and we expect the apo BCCP would have a similar effect on BirA self modification in vivo. Indeed, a wild type E. coli cell contains only about ten or so molecules of BirA and BCCP is normally present in great excess.…”

Section: Discussionmentioning

confidence: 89%

“…The observed self modification was not unexpected, since two other enzymes that synthesize acyl-adenylate intermediates, the methionyl-tRNA synthetases of E. coli and Bacillus stearothermophilus, have been shown to catalyze self methionylation (Gillet et al 1997;Hountondji et al 2000). In these enzymes it was reported that once methionine was activated by ATP to form methionine-5Ј-AMP, the reactive lysine side chains closest to the acyl-adenylate were those modified (Hountondji et al 2000).…”

Section: Discussionmentioning

confidence: 92%

Promiscuous protein biotinylation by Escherichia coli biotin protein ligase

2004

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Biotin protein ligases (BPLs) are enzymes of extraordinary specificity. BirA, the BPL of Escherichia coli biotinylates only a single cellular protein. We report a mutant BirA that attaches biotin to a large number of cellular proteins in vivo and to bovine serum albumin, chloramphenicol acetyltransferase, immunoglobin heavy and light chains, and RNAse A in vitro. The mutant BirA also self biotinylates in vivo and in vitro. The wild type BirA protein is much less active in these reactions. The biotinylation reaction is proximitydependent in that a greater extent of biotinylation was seen when the mutant ligase was coupled to the acceptor proteins than when the acceptors were free in solution. This approach may permit facile detection and recovery of interacting proteins by existing avidin/streptavidin technology.Keywords: biotin protein ligase; protein modification; biotinylation; acyl adenylate; BirA Biotinylation of proteins has routinely been done by chemical means, usually by modification of protein amino groups with biotin-N-hydroxysuccinimide or similar acylating agents. In contrast, enzymatic biotinylation has been limited to the few proteins that normally carry this modification, which are largely biotin-dependent carboxylases and decarboxylases of central metabolism (Chapman-Smith and Cronan Jr. 1999). We supposed that if enzymatic biotinylation could be made less specific, it might provide a means to detect weak (i.e., having dissociation constants >10 −7 M) protein-protein interactions. In this scenario the biotinylating enzyme physically coupled to one of the interacting proteins (the target protein) would be used to biotinylate and thereby tag proteins that interact with the target protein. The biotinylation reaction should tag only those protein molecules that are close in space to the target protein. That is, unlike chemical acylation, enzymatic biotinylation should be proximity-dependent. The specific and extremely tight binding of biotin (K D 10 −13 to 10 −15 M) to streptavidin and avidin would then allow very sensitive detection of biotinylated proteins by a wide variety of robust protocols and low affinity forms of streptavidin and avidin allow efficient purification of biotinylated proteins under mild conditions. However, the biotin protein ligases (BPLs) catalyzing protein biotinylation have exceptional specificities for their protein substrates and thus are not general protein modification enzymes. For example, in vivo, BirA, the BPL of Escherichia coli, biotinylates only a single protein, the BCCP (AccB) subunit of acetyl-CoA carboxylase and other organisms contain less than five biotinylated protein species, indicating these BPLs are similarly specific (McAllister and Coon 1966;Samols et al. 1988;. Therefore, in order to use a BPL as a general biotinylating enzyme, this extraordinary specificity must somehow be overcome. A possible route to this end is based upon the mechanism of the BPL reaction, which proceeds in two steps:1. Biotin + ATP Bio-5Ј-AMP + PPi 2. Bio-5Ј-AMP + apo-Prot...

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“…Troubleshooting 3.4.1 Acid gel electrophoresis to confirm the location of the mischarged amino acid-Protein co-precipitates with tRNA during TCA precipitation (Section 3.3). Under conditions where the enzyme's charging activity is weak, such as during mischarging, it is possible for the enzyme to become self-labeled with radiolabeled amino acids (72)(73)(74). In addition, radiolabeled amino acid may be trapped in the protein's active site and also coprecipitated.…”

Section: Assay To Measure Deacylation Of Ala-trna Promentioning

confidence: 99%

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

Splan

Musier‐Forsyth

Boniecki

et al. 2008

Methods

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Aminoacyl-tRNA synthetases are essential enzymes that help to ensure the fidelity of protein translation by accurately aminoacylating (or "charging") specific tRNA substrates with cognate amino acids. Many synthetases have an additional catalytic activity to confer amino acid editing or proofreading. This activity relieves ambiguities during translation of the genetic code that result from one synthetase activating multiple amino acid substrates. In this review, we describe methods that have been developed for assaying both pre-and post-transfer editing activities. Pre-transfer editing is defined as hydrolysis of a misactivated aminoacyl-adenylate prior to transfer to the tRNA. This reaction has been reported to occur either in the aminoacylation active site or in a separate editing domain. Post-transfer editing refers to the hydrolysis reaction that cleaves the aminoacyl-ester linkage formed between the carbonyl carbon of the amino acid and the 2′ or 3′ hydroxyl group of the ribose on the terminal adenosine. Post-transfer editing takes place in a hydrolytic active site that is distinct from the site of amino acid activation. Here, we focus on methods for determination of steady-state reaction rates using editing assays developed for both classes of synthetases.

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Covalent methionylation of escherichia coli methionyl‐tRNA synthethase: Identification of the labeled amino acid residues by matrix‐assisted laser desorption‐ionization mass spectrometry

Cited by 18 publications

References 46 publications

Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Adaptation to tRNA acceptor stem structure by flexible adjustment in the catalytic domain of class I tRNA synthetases

Promiscuous protein biotinylation by Escherichia coli biotin protein ligase

In vitro assays for the determination of aminoacyl-tRNA synthetase editing activity

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