title: 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-tRNAPro. The most important functional element of this catalytic mechanism is the 2'-OH group of the terminal adenosine 76 of Ala-tRNAPro, 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.
authors.affiliation: a Department of Protein Synthesis Enzymology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.|b State Key Laboratory of Molecular and Cellular Biology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.
authors.forename: Konstantin S
authors.initials: KS
authors.lastname: Boyarshin
authors.affiliation: b State Key Laboratory of Molecular and Cellular Biology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.
authors.forename: Anastasia E
authors.initials: AE
authors.lastname: Priss
authors.affiliation: a Department of Protein Synthesis Enzymology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.|b State Key Laboratory of Molecular and Cellular Biology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.
authors.forename: Alexsey V
authors.initials: AV
authors.lastname: Rayevskiy
authors.affiliation: c Department of Synthetic Bioregulators , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.
authors.forename: Mykola M
authors.initials: MM
authors.lastname: Ilchenko
authors.affiliation: c Department of Synthetic Bioregulators , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.
authors.forename: Igor Ya
authors.initials: IY
authors.lastname: Dubey
authors.affiliation: a Department of Protein Synthesis Enzymology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.|b State Key Laboratory of Molecular and Cellular Biology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.
authors.forename: Ivan A
authors.initials: IA
authors.lastname: Kriklivyi
authors.affiliation: a Department of Protein Synthesis Enzymology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.|b State Key Laboratory of Molecular and Cellular Biology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.
authors.forename: Anna D
authors.initials: AD
authors.lastname: Yaremchuk
authors.affiliation: a Department of Protein Synthesis Enzymology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.|b State Key Laboratory of Molecular and Cellular Biology , Institute of Molecular Biology and Genetics, National Academy of Sciences of Ukraine , 150 Zabolotnogo Str, 03680 Kyiv , Ukraine.
authors.forename: Michael A
authors.initials: MA
authors.lastname: Tukalo
mesh_terms: D000604:Amino Acyl-tRNA Synthetases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D001419:Bacteria / Q000201:enzymology / Q000235:genetics; D020134:Catalytic Domain; D006860:Hydrogen Bonding; D006868:Hydrolysis; D008958:Models, Molecular; D008968:Molecular Conformation; D011485:Protein Binding; D012343:RNA, Transfer / Q000737:chemistry* / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D012343:RNA, Transfer; D000604:Amino Acyl-tRNA Synthetases; C100134:prolyl T RNA synthetase
keywords: MD and QM methods; aminoacyl-tRNA synthetase; post-transfer editing mechanism; prolyl-tRNA synthetase; tRNA