Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry
Soria Baouz#1, Jean-Marie Schmitter#2, Lila Chenoune1, Christian Beauvallet3, Sylvain Blanquet4, Anne Woisard1, Codjo Hountondji*, 1
1 UPMC Univ Paris 06 et Univ Paris 13, Institut Jacques Monod (Tour 43) Laboratoire de Photobiologie Moléculaire (CNRS-UMR 7033, BioMoCeTi), Case courrier 7592 - 2, Place Jussieu, F-75251, Paris, Cedex 05 France
2 Institut Européen de Chimie et Biologie (CNRS-FRE 2247), F-33607, Pessac, France
3 Institut National de la Recherche Agronomique (INRA GPL), F-78352, Jouy-en-Josas, France
4 Laboratoire de Biochimie (CNRS UMR 7654), Ecole Polytechnique, Route de Saclay, F-91128, Palaiseau, France
The correct amino acid sequence of E. coli isoleucyl-tRNA synthetase (IleRS) was established by means of peptide mapping by MALDI mass spectrometry, using a set of four endoproteases (trypsin, LysC, AspN and GluC). Thereafter, the active site of IleRS was mapped by affinity labeling with reactive analogs of the substrates. For the ATP binding site, the affinity labeling reagent was pyridoxal 5'-diphospho-5'-adenosine (ADP-PL), whereas periodate-oxidized tRNAIle, the 2',3'-dialdehyde derivative of tRNAIle was used to label the binding site for the 3'-end of tRNA on the synthetase. Incubation of either reagent with IleRS resulted in a rapid loss of both the tRNAIle aminoacylation and isoleucinedependent isotopic ATP-PPi exchange activities. The stoichiometries of IleRS labeling by ADP-PL or tRNAIleox corresponded to 1 mol of reagent incorporated per mol of enzyme. Altogether, the oxidized 3'-end of tRNAIle and the pyridoxal moiety of the ATP analog ADP-PL react with the lysyl residues 601 and 604 of the consensus sequence 601KMSKS605. Identification of the binding site for L-isoleucine or for non cognate amino acids on E. coli IleRS was achieved by qualitative comparative labeling of the synthetase with bromomethyl ketone derivatives of L-isoleucine (IBMK) or of the non-cognate amino acids valine (VBMK), phenylalanine (FBMK) and norleucine (NleBMK). Labeling of the enzyme with IBMK resulted in a complete loss of isoleucine-dependent isotopic [32P]PPi-ATP exchange activity. VBMK, NleBMK and FBMK were also capable of abolishing the activity of IleRS, FBMK being the less efficient in inactivating the synthetase. Analysis by MALDI mass spectrometry designated cysteines-462 and -718 as the target residues of the substrate analog IBMK on E. coli IleRS, whereas VBMK, NleBMK and FBMK labeled in common His-394, His-478 and Cys-718. In addition, VBMK and NleBMK, which are chemically similar to IBMK, were found covalently bound to Cys-462, and VBMK was specifically attached to His-332 (or His-337) of the synthetase. The amino acid residues labeled by the substrate analogs are mainly distributed between three regions in the primary structure of E. coli IleRS: these are segments [325-394], [451-479] and [591-604]. In the 3-D structures of IleRS from T. thermophilus and S. aureus, the [325-394] stretch is part of the editing domain, while fragments [451-479] and [591-604] representing the isoleucine binding domain and the dinucleotide (or Rossmann) fold domain, respectively, are located in the catalytic core. His-332 of E. coli IleRS, that is strictly conserved among all the available IleRS sequences is located in the editing active site of the synthetase. It is proposed that His-332 of E. coli IleRS participates directly in hydrolysis, or helps to deprotonate the hydroxyl group of threonine at the hydrolytic site.
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* Address correspondence to this author at the Université Pierre et Marie Curie (UPMC), Laboratoire de Photobiologie Moléculaire, (UMR-CNRS 7033, BioMoCeTi) – Institut Jacques Monod (Tour 43) – Case Courrier N° 7592 – 2, Place Jussieu, 75251 Paris Cedex 05, France; Tel: (33) (0)188.8.131.52.86. Fax: (33) (0)1. 184.108.40.206; E-mail: firstname.lastname@example.org#These authors contributed equally to this work.