Valyl‐tRNA synthetase from Escherichia coli

Abstract

Bromomethyl ketone derivatives of l-valine (VBMK), l-isoleucine (IBMK), l-norleucine (NleBMK) and l-phenylalanine (FBMK) were synthesized. These reagents were used for qualitative comparative labeling of Escherichia coli valyl-tRNA synthetase (ValRS), an enzyme with Val/Ile editing activity, in order to identify the binding sites for l-valine or noncognate amino acids. Labeling of E. coli ValRS with the substrate analog valyl-bromomethyl ketone (VBMK) resulted in a complete loss of valine-dependent isotopic [ 32 P]PPi±ATP exchange activity. l-Valine protected the enzyme against inactivation. Noncognate amino acids analogs isoleucyl-, norleucyl-and phenylalanyl-bromomethyl ketones (IBMK, NleBMK and FBMK) were also capable of abolishing the activity of ValRS, FBMK being less efficient in inactivating the synthetase. Matrix-assisted laser desorption-ionization mass spectrometry designated cysteines 424 and 829 as the target residues of the substrate analog VBMK on E. coli ValRS, whereas, altogether, IBMK, NleBMK and FBMK labeled His266, Cys275, His282, His433 and Cys829, of which Cys275, His282 and His433 were labeled in common by all three noncognate amino-acid-derived bromomethyl ketones. With the exception of Cys829, which was most likely unspecifically labeled, the amino-acid residues labeled by the reagents derived from noncognate amino acids were distributed between two fragments 259±291 and 419±434 in the primary structure of E. coli ValRS. In fragment 419±434, Cys424 was specifically labeled by the substrate analog VBMK, while His433 was labeled in common by all the used bromomethyl ketone derivatives of noncognate amino acids, suggesting that the synthetic site where aminoacyl adenylate formation takes place on E. coli ValRS is built up of two subsites. One subsite containing Cys424 might represent the catalytic locus of the active center where specific l-valine activation takes place. The second subsite containing His433 might represent the binding site for noncognate amino acids. The fact that Cys275 and His282, fragment 259±291, were labeled by IBMK, NleBMK and FBMK, but not by the substrate analog VBMK, suggests that these residues might be located at or near the editing site of E. coli ValRS. Comparison of fragment 259±291 with all the available ValRS amino-acid sequences revealed that His282 is strictly conserved, with the exception of its replacement by a glycine in a subgroup corresponding to the archaebacteria. Because a nucleophile is needed in the editing site to achieve hydrolysis of an undesired product at the level of the carbonyl group thereof, it is proposed that the conserved His282 of E. coli ValRS is involved in editing.Keywords: amino-acid binding site; editing site; MALDI-MS; valyl-tRNA synthetase.During protein biosynthesis, accurate reading of the genetic code is determined by two key functions of aminoacyl-tRNA synthetases. First, each of the 20 or so synthetases correctly matches a cognate amino acid with a cognate tRNA. The precision with which these enzymes catalyze the overall a...

The Crystal Structure of E. coli Pantothenate Synthetase Confirms It as a Member of the Cytidylyltransferase Superfamily

“…Both of density reconstruction using BUSTER [18], whereas native and selenomethionine-substituted (SeMet) enresidues B63-68 and A-B251-259 have temperature faczyme were purified using the procedure described in tors approaching 2); this is also the case between the various tRS sequences themselves. However, the two sequences from both plants and microorganisms, which share about 50% pairwise similarity to one another over sequence motifs that have previously been noted in tRS, HIGH [10] and KMSKS [29], are boxed in Figure 3c; the the entire protein [8]. In all the known plant sequences, the dimerization domain is preceded by an insertion of HIGH motif binds the adenine portion of ATP (cytidine in CGT), whereas the KSMKS motif stabilizes the ␤and about 20 amino acids (between residues 99 and 100 relative to the E. coli sequence).…”

Primary Structure Revision and Active Site Mapping of E. Coli Isoleucyl-tRNA Synthetase by Means of Maldi Mass Spectrometry

“…In a previous work, we have used affinity labeling [12] and protein modification [38] studies to identify amino acid residues at the synthetic and the hydrolytic editing active sites on E. coli ValRS. Among the amino acid residues of the editing active site of this synthetase, Lys-277, a residue strictly conserved from bacteria to human, corresponds to His-332 of E. coli IleRS [38] which plays a crucial role in editing ([37] and this work).…”

“…However, the nucleophile His-319 of Thermus thermophilus IleRS is not conserved in the ValRS and LeuRS amino acid sequences, suggesting that the involvement of a mechanism analogous to the charge relay system in the editing site of the ValRS, IleRS and LeuRS families is less probable. On the bases of the data of the present work on Escherichia coli IleRS, and of the qualitative comparative labeling of Escherichia coli ValRS with reactive amino acids analogs [12, 38], we propose that the role of nucleophilic residues in the editing active site of the synthetases consists of merely helping to deprotonate the catalytic editing residues, in order to make them capable of attacking the carbonyl group of the ester of misactivated or misacylated non cognate amino acids. This hypothesis is supported by the observation by Edsall and Wyman [43], that situation of a nucleophile at a two-residue distance from another one may increase the nucleophilic character of each of them, by lowering the pKa values by up to 2 pH units.…”

“…Bromomethyl ketone derivatives of amino acids were synthesized as already described [12]. Isoleucyl-tRNA synthetase (IleRS), stored in 20 mM Tris-HCl buffer (pH 7.8) containing 50% glycerol and 10 mM 2-mercaptoethanol, was extensively dialyzed against the appropriate buffer prior to incubation with a given reagent.…”

“…Pyridoxal 5'-phosphate and pyridoxal 5'-diphosphate have also been used to identify the subsite for the pyrophosphate moiety or for the γ-phosphate of ATP [10, 11]. Nucleophilic amino acid residues at the binding site for L-valine or for non-cognate amino acids were identified by comparative labeling of valyl-tRNA synthetase (ValRS) with bromomethylketone derivatives [12]. The latter site referred to as the editing site is responsible for the Val/Thr editing activity of E. coli ValRS which prevents attachment of L-threonine to tRNA Val [13].…”