Aminoacyl tRNA synthetases are enzymes that specifically attach amino acids to cognate tRNAs for use in the ribosomal stage of translation. For many aminoacyl tRNA synthetases, the required level of amino acid specificity is achieved either by specific hydrolysis of misactivated aminoacyl-adenylate intermediate (pre-transfer editing) or by hydrolysis of the mischarged aminoacyl-tRNA (post-transfer editing). To investigate the mechanism of post-transfer editing of alanine by prolyl-tRNA synthetase from the pathogenic bacteria Enterococcus faecalis, we used molecular modeling, molecular dynamic simulations, quantum mechanical (QM) calculations, site-directed mutagenesis of the enzyme, and tRNA modification. The results support a new tRNA-assisted mechanism of hydrolysis of misacylated Ala-tRNA. The most important functional element of this catalytic mechanism is the 2'-OH group of the terminal adenosine 76 of Ala-tRNA, which forms an intramolecular hydrogen bond with the carbonyl group of the alanine residue, strongly facilitating hydrolysis. Hydrolysis was shown by QM methods to proceed via a general acid-base catalysis mechanism involving two functionally distinct water molecules. The transition state of the reaction was identified. Amino acid residues of the editing active site participate in the coordination of substrate and both attacking and assisting water molecules, performing the proton transfer to the 3'-O atom of A76.

The maintenance of amino acid specificity by aminoacyl-tRNA synthetases can require the hydrolysis of missynthesized products that is known as amino acid editing. Bacterial prolyl-tRNA synthetase includes a special editing domain, that deacylates alanyl-tRNAPro, and so exhibits post-transfer editing activity. The mechanism of tRNA-dependent editing by prolyl-tRNA synthetase has to be defined. The present work aim is to study the structure of the active site of enterobacteria E. faecalis prolyl-tRNA synthetase editing domain. The amino acids positions E218, T257, K279, G331, S332, G334, and H366 have been chosen for the site-directed mutagenesis (alanine scanning). An editing activity of the mutants was compared with the wild type prolyl-tRNA synthetase. Three amino acid residues, important for the editing activity, K279, G331 and H366, were revealed. This data are consistent with the existing suppositions about the structure of bacterial prolyl-tRNA synthetase deacylating active sit

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Aim. Cloning, sequencing and expression of the gene encoding prolyl-tRNA synthetase, a class IIa enzyme, from the extreme thermophile T. thermophilus HB8 (ProRSTT). Methods. Search for the ProRSTT gene was performed by Southern blot hybridization with chromosomal DNA, the digoxigenin-labeled PCR fragments of DNA being used as a probe. Results. The gene of T. thermophilus HB8 ProRS has been cloned and sequenced. The predicted 477-amino acid protein is significantly more similar in sequence to eukaryotic and archaeal than to eubacterial ProRS. Sequence analysis shows two distinct structural groups of ProRS which most likely had diverged early in evolution: (1) eukaryotic/archaeal group characterized by the absence of an insertion domain between motifs 2 and 3 and by the presence of an extra C-terminal domain beyond the normal class IIa anticodon binding domain; and (2) prokaryotic with a very large insertion between motif 2 and 3 and no extra C-terminal domain. Conclusions. T. thermophilus proS gene was overexpressed in Escherichia coli and overproduced ProRSTT was purified to high homogeneity. In spite of its eukaryotic-like features, T. thermophilus ProRS exhibited highly specific cross-species aminoacylation. The charging ability of the ProRSTT is restricted to prokaryotic tRNAPro