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

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.

Keywords: acetylation; amino acid; aminoacyl tRNA synthetase; catalysis; transfer RNA (tRNA); translation.

Figure 1.

Identification of acetylation at Lys⁶¹⁹ of EcLeuRS by MS. MS/MS spectrum of a tryptic peptide from EcLeuRS (DAAGHELVYTGMSK^AcMSK) shows acetylation of Lys (K^Ac), confirmed as Lys⁶¹⁹ by sequence alignment with the known sequence of EcLeuRS. Most major fragmentation ions matched predicted b or y ions.

Figure 2.

Identification of Lys residues acetylated in EcLeuRS. A, the overall ternary structure of EcLeuRS and its cognate tRNA^Leu together with Leu in the editing conformation (PDB number 4ARC). B, schematic diagram of EcLeuRS. RF, Rossmann fold. C, aminoacylation assays screening potential crucial Lys residues. Left panel, mutation of Lys⁶¹⁹, Lys⁶²⁴, and Lys⁸⁰⁹ to Gln damaged EcLeuRS canonical activities. Right panel, mutation of other Lys residues had a slightly negative effect on EcLeuRS canonical activities. The results are averages plus standard deviations from three independent experiments.

Figure 3.

Flow diagram of the overexpression of site-directed AcK-incorporated proteins in E. coli BL21 (DE3). Taking EcLeuRS as an example, pAcKRS and pET22b(+)-ecleuS were co-transformed in E. coli BL21 (DE3), and engineered pAcKRS were induced by the addition of Ara. Subsequently, IPTG was added to induce the production of EcLeuRS in the presence of NAM, an inhibitor of CobB. With the assistance of pAcKRS, EcLeuRS was translated in full-length form with incorporation of AcK or in truncated form (terminating at the Lys codon mutation site). All other experimental details were as described previously (43).

Figure 4.

Effect of acetylation of Lys residues on the Leu activation and aminoacylation activities of EcLeuRS. A, sequence alignment of LeuRSs from various species in regions homologous to Lys⁶¹⁹, Lys⁶²⁴, and Lys⁸⁰⁹ in EcLeuRS. Ec, E. coli; Aa, Aquifex aeolicus; Bs, B. subtilis; Sco, Streptomyces coelicolor; Hs, H. sapiens; Ph, Pyrococcus horikoshii; mt, mitochondrial; ct, cytoplasmic. B, Western blotting confirming the incorporation of AcK in EcLeuRS-K619^Ac, EcLeuRS-K624^Ac, and EcLeuRS-K809^Ac. C, Leu activation of EcLeuRS-K619^Ac, EcLeuRS-K624^Ac, and EcLeuRS-K809^Ac. D, aminoacylation of EcLeuRS-K619^Ac, EcLeuRS-K624^Ac, and EcLeuRS-K809^Ac resembling that of the K-Q mutants. E, closer view of the orientation of Lys⁶¹⁹ and Lys⁶²⁴ relative to the conserved HIGH and KMSK motifs (HMGH and KMSK in EcLeuRS, depicted in dark blue; PDB code 4ARC). F, closer view of the interaction between EcLeuRS Lys⁸⁰⁹ and EctRNA^Leu U47I (PDB code 4ARC). The results are the averages and standard deviations from three independent experiments, and all Western blots were repeated.

Figure 5.

AcP and CobB regulate acetylation of EcLeuRS. A, AcP but not YfiQ acetylates EcLeuRS in vitro. B, CobB removal of the acetyl moiety of EcLeuRS^Ac. C, CobB deacetylation of EcLeuRS-K619^Ac and EcLeuRS-K809^Ac. In the presence of NAM or the absence of NAD⁺, CobB is inactivated. D, incubation with CobB recovers the aminoacylation activity of EcLeuRS-K619^Ac (left panel) and EcLeuRS-K809^Ac (right panel). All experiments were conducted at least twice. When quantifying of the relative amount of AcK signal/His signal, the sample without NAD⁺ and NAM was defined as 100%.

Figure 6.

Identification of acetylation at Lys¹²⁶ of EcArgRS by MS. MS/MS spectrum of a tryptic peptide from EcArgRS (QTIVVDYSAPNVAK^AcEMHVGHLR) showing acetylation of Lys (K^Ac), confirmed as Lys¹²⁶ by sequence alignment of the peptide with the known sequence of EcArgRS. Most major fragmentation ions matched predicted b or y ions.

Figure 7.

Lys residues acetylated in EcArgRS. A, crystal structure of PhArgRS complexed with PhtRNA^Arg and ANP (PDB code 2ZUE). B, schematic diagram of EcArgRS. RF, Rossmann fold. C, aminoacylation assay screening of potentially crucial Lys residues. Left panel, mutation of Lys¹²⁶ and Lys⁴⁰⁸ damages the enzymatic activities of EcArgRS. Right panel, mutation of other Lys residues has a slight negative effect on the activities EcArgRS. The results are the averages and standard deviations from three independent experiments.

Figure 8.

Effect of acetylation of Lys on the Leu activation and aminoacylation activities of EcArgRS. A, sequence alignment of ArgRSs from various species in regions homologous to Lys¹²⁶ and Lys⁴⁰⁸ of EcArgRS. The abbreviations are the same as those in Fig. 4, except for two species (Se, S. enterica; Sc, S. cerevisiae). B, Arg activation of EcArgRS-K^Acs. C, aminoacylation of EcArgRS-K^Acs resembling that of the K-Q mutants. D, orientation of PhArgRS Lys¹³² (homologous to Lys¹²⁶ in EcArgRS) relative to the conserved HIGH motif (HMGH in PhArgRS, depicted in dark blue). E, closer view of the interaction between PhArgRS Lys⁴⁵⁵ (corresponding to Lys⁴⁰⁸ in EcArgRS) and PhtRNA^Arg A38. The results are the averages and standard deviations from three independent experiments.

Figure 9.

AcP and CobB regulate acetylation of EcArgRS. A, AcP acetylates EcArgRS in vitro. B, CobB removes the acetyl moiety of EcArgRS^Ac. C, CobB deacetylates EcArgRS-K126^Ac and other acetylated variants. CobB deacetylation activity is lost in the presence of NAM or the absence of NAD⁺. All experiments were performed at least twice. When quantifying of the relative amount of AcK signal/His signal, the group without NAD⁺ and NAM was defined as 100%.

Figure 10.

Sequence alignment of the HIGH region and the acetate metabolism pathway. A, sequence alignment of the HIGH region with ArgRSs from various species. Lys residues preceding the HIGH motif (indicated by an arrow, Lys¹²⁶ in EcArgRS) were found to be acetylated by MS. Abbreviations are the same as Figs. 4 and 8, except for two species (Rn, R. norvegicus; Mm, M. musculus). B, acetate metabolism pathway in E. coli (23). Pta, AckA, and Acs are crucial enzymes involved in the interconversion of Ac-CoA and acetate.

Figure 11.

Proposed acetylation mechanism for aaRSs. In this model, AcP nonenzymatically acetylates aaRSs, which negatively regulates their aminoacylation activities, and this PTM is removed by CobB to recover aaRS function and maintain cellular homeostasis (29).