Acetylation of lysine ϵ-amino groups regulates aminoacyl-tRNA synthetase activity in Escherichia coli

Qing Ye,Quan-Quan Ji,Wei Yan,Fang Yang,En-Duo Wang

doi:10.1074/jbc.m116.770826

Qing Ye, Quan-Quan Ji + Show 3 more

Open Access

https://doi.org/10.1074/jbc.m116.770826

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Abstract

Previous proteomic analyses have shown that aminoacyl-tRNA synthetases in many organisms can be modified by acetylation of Lys. In this present study, leucyl-tRNA synthetase and arginyl-tRNA synthetase from Escherichia coli (EcLeuRS and EcArgRS) were overexpressed and purified and found to be acetylated on Lys residues by MS. Gln scanning mutagenesis revealed that Lys619, Lys624, and Lys809 in EcLeuRS and Lys126 and Lys408 in EcArgRS might play important roles in enzyme activity. Furthermore, we utilized a novel protein expression system to obtain enzymes harboring acetylated Lys at specific sites and investigated their catalytic activity. Acetylation of these Lys residues could affect their aminoacylation activity by influencing amino acid activation and/or the affinity for tRNA. In vitro assays showed that acetyl-phosphate nonenzymatically acetylates EcLeuRS and EcArgRS and suggested that the sirtuin class deacetylase CobB might regulate acetylation of these two enzymes. These findings imply a potential regulatory role for Lys acetylation in controlling the activity of aminoacyl-tRNA synthetases and thus protein synthesis.

Highlights

Aminoacyl-tRNA synthetase2 catalyzes esterification between its cognate amino acid and tRNA to produce aminoacyl-tRNA in the initiation step of translation
To understand the effect of acetylation on the aminoacylation activity of EcLeuRS, we separately mutated all 11 Lys residues to Gln, because this residue lacks a positive charge on the side chain and is a good mimic of acetylated Lys
Lys402 is the only residue in the connective peptide 1 (CP1) domain among these 11 residues, and the co-crystal structure of EcLeuRS with tRNALeu and Leu (PDB number 4ARC) indicated that Lys402 lies on the surface of the CP1 pocket and

Summary

Introduction

Aminoacyl-tRNA synthetase (aaRS) catalyzes esterification between its cognate amino acid and tRNA to produce aminoacyl-tRNA (aa-tRNA) in the initiation step of translation. (HIGH and KMSK) located in the active site that form a characteristic dinucleotide binding fold (Rossmann fold, ␤-␣-␤-␣␤). Both leucyl- and arginyl-tRNA synthetases (LeuRS and ArgRS) belong to class I aaRSs [5]. It is peculiar that the Add domain is conserved in ArgRS but not in other class I aaRSs. In most species, ArgRS lacks a canonical KMSK sequence, and a conserved lysine (Lys) upstream of the HIGH sequence motif in these enzymes stabilizes the transition state of the amino acid activation reaction (Arg-AMP formation) to compensate for the loss of the second Lys (K2) in the KMSK motif [16, 17]

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Discussion

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