A new mechanism of post-transfer editing by aminoacyl-tRNA synthetases: catalysis of hydrolytic reaction by bacterial-type prolyl-tRNA synthetase

Abstract

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.

Computational approaches for parameterization of aminoacyl-tRNA synthetase substrates

“…However, the most accurate and illustrative estimation of the model is [an] implementation of the newborn topology in the MD simulation. , which confirm[s] a better compliance of RESP model with experimental data, described in the paper [10]. It is evident that a decrease of electrostatic interaction energy value during the MD is caused by an incorrect turn of alanine radical and loss of its hydrogen bonds and disappearance of water molecules (sterical repulsion), which are responsible for nucleophilic attack and hydrolysis.…”

“…From the Figure it becomes evident that RESP charge gives much more accurate and reliable results, which correlate with the experimental data [10]. The molecular dynamics of ProRS system also aimed at investigation of the 2'OH group impact and showed the conformity between biochemical study and computational calculations (not shown here).…”

“…The protein structure was prepared and reconstruct-ed on the basis of the deposited X-ray (2J3M) to rebuild a full-length structure without gaps, using Swiss-model server [9]. The procedure of ligand-protein and nucleic acid-protein docking protocol has been already highlighted in the previous study [10]. A significant attention was paid to the torsion angle "CT-OS-C" of the aminoacyl-tRNA molecule, which is not a part of the AMBER99 force field but plays an important role in an excessive flexibility of the molecule during MD.…”

“…To study the molecular mechanism of the editing reaction, the structure of ProRS from E.faecalis in complex with tRNA Pro was remodeled and validated in appropriate way [10]. We studied a prolyl-tRNA synthetase (II class) system, which possess[es] an editing activity against misactivated alanyl-tRNA via INS domain.…”

“…Our model was used for the QM calculations of all predictable reaction mechanisms. The approach used allowed us to perform the direct identification of the transition state of the reaction pathway and to support a new mechanism of hydrolysis of misacylated Ala-tRNA Pro [10].…”