Article

Inhibition by L-aspartol adenylate of a nondiscriminating aspartyl-tRNA synthetase reveals differences between the interactions of its active site with tRNA(Asp) and tRNA(Asn).

Centre de Recherche en Infectiologie, CHUL, 2705 Boul. Laurier RC-709, Québec, QC, Canada.
Journal of Enzyme Inhibition and Medicinal Chemistry (impact factor: 1.62). 03/2007; 22(1):77-82. DOI:10.1080/14756360600952316 pp.77-82
Source: PubMed

ABSTRACT Asparaginyl-tRNA formation in Pseudomonas aeruginosa PAO1 involves a nondiscriminating aspartyl-tRNA synthetase (ND-AspRS) which forms Asp-tRNA(Asp) and Asp-tRNA(Asn), and a tRNA-dependent amidotransferase which transamidates the latter into Asn-tRNA(Asn). We report here that the inhibition of this ND-AspRS by L-aspartol adenylate (Asp-ol-AMP), a stable analog of the natural reaction intermediate L-aspartyl adenylate, is biphasic because the aspartylation of the two tRNA substrates of ND-AspRS, tRNA(Asp) and tRNA(Asn), are inhibited with different Ki values (41 microM and 215 microM, respectively). These results reveal that the two tRNA substrates of ND-AspRS interact differently with its active site. Yeast tRNA(Asp) transcripts with some identity elements replaced by those of tRNA(Asn) have their aspartylation inhibited with Ki values different from that for the wild-type transcript. Therefore, aminoacyl adenylate analogs, which are competitive inhibitors of their cognate aminoacyl-tRNA synthetase, can be used to probe rapidly the role of various structural elements in positioning the tRNA acceptor end in the active site.

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    Article: Peculiar inhibition of human mitochondrial aspartyl-tRNA synthetase by adenylate analogs.
    Marie Messmer, Sébastien P Blais, Christian Balg, Robert Chênevert, Luc Grenier, Patrick Lagüe, Claude Sauter, Marie Sissler, Richard Giegé, Jacques Lapointe, Catherine Florentz
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    Biochimie 03/2009; 91(5):596-603. · 3.02 Impact Factor
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    Article: The asparagine-transamidosome from Helicobacter pylori: a dual-kinetic mode in non-discriminating aspartyl-tRNA synthetase safeguards the genetic code.
    Frédéric Fischer, Jonathan L Huot, Bernard Lorber, Guillaume Diss, Tamara L Hendrickson, Hubert D Becker, Jacques Lapointe, Daniel Kern
    [show abstract] [hide abstract]
    ABSTRACT: Helicobacter pylori catalyzes Asn-tRNA(Asn) formation by use of the indirect pathway that involves charging of Asp onto tRNA(Asn) by a non-discriminating aspartyl-tRNA synthetase (ND-AspRS), followed by conversion of the mischarged Asp into Asn by the GatCAB amidotransferase. We show that the partners of asparaginylation assemble into a dynamic Asn-transamidosome, which uses a different strategy than the Gln-transamidosome to prevent the release of the mischarged aminoacyl-tRNA intermediate. The complex is described by gel-filtration, dynamic light scattering and kinetic measurements. Two strategies for asparaginylation are shown: (i) tRNA(Asn) binds GatCAB first, allowing aminoacylation and immediate transamidation once ND-AspRS joins the complex; (ii) tRNA(Asn) is bound by ND-AspRS which releases the Asp-tRNA(Asn) product much slower than the cognate Asp-tRNA(Asp); this kinetic peculiarity allows GatCAB to bind and transamidate Asp-tRNA(Asn) before its release by the ND-AspRS. These results are discussed in the context of the interrelation between the Asn and Gln-transamidosomes which use the same GatCAB in H. pylori, and shed light on a kinetic mechanism that ensures faithful codon reassignment for Asn.
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Keywords

41 microM
 
aminoacyl adenylate analogs
 
Asparaginyl-tRNA formation
 
biphasic
 
cognate aminoacyl-tRNA synthetase
 
competitive inhibitors
 
different Ki values
 
Ki values different
 
L-aspartol adenylate
 
natural reaction intermediate L-aspartyl adenylate
 
ND-AspRS
 
ND-AspRS interact
 
nondiscriminating aspartyl-tRNA synthetase
 
positioning
 
Pseudomonas aeruginosa PAO1
 
stable analog
 
tRNA-dependent amidotransferase
 
wild-type transcript