Abstract
Protein lysine acetylation is a widely conserved posttranslational modification in all three domains of life. Lysine acetylation frequently occurs in aminoacyl-tRNA synthetases (aaRSs) from many organisms. In this study, we determined the impact of the naturally occurring acetylation at lysine-73 (K73) in Escherichia coli class II alanyl-tRNA synthetase (AlaRS) on its alanylation activity. We prepared an AlaRS K73Ac variant in which Nε-acetyl-l-lysine was incorporated at position 73 using an expanded genetic code system in E. coli. The AlaRS K73Ac variant showed low activity compared to the AlaRS wild type (WT). Nicotinamide treatment or CobB-deletion in an E. coli led to elevated acetylation levels of AlaRS K73Ac and strongly reduced alanylation activities. We assumed that alanylation by AlaRS is affected by K73 acetylation, and the modification is sensitive to CobB deacetylase in vivo. We also showed that E. coli expresses two CobB isoforms (CobB-L and CobB-S) in vivo. CobB-S displayed the deacetylase activity of the AlaRS K73Ac variant in vitro. Our results imply a potential regulatory role for lysine acetylation in controlling the activity of aaRSs and protein synthesis.
Highlights
The recent development of mass spectrometry (MS)-based proteomic analysis enables the discovery of a wide variety of posttranslational modifications (PTMs) in prokaryotes [1]
We demonstrate that one of the class II aminoacyl-tRNA synthetases (aaRSs), alanyl-tRNA synthetase (AlaRS)
K73 is an essential residue in motif of the enzyme active site
Summary
The recent development of mass spectrometry (MS)-based proteomic analysis enables the discovery of a wide variety of posttranslational modifications (PTMs) in prokaryotes [1]. Proteomic studies of acetylated protein (acetylome) indicated a large number of acetylated proteins in bacteria and archaea [2,3,4,5,6,7]. Since they possess homologs of eukaryotic lysine acetyltransferases (KATs) and deacetylases (KDACs) [8], acetylation in bacteria and archaea are expected to be involved in the regulation of cellular processes as in eukaryotes. In Escherichia coli, acetylation is introduced to lysine residues by a KAT-dependent reaction utilizing acetyl-CoA and a non-enzymatic reaction utilizing acetyl-phosphate [15,16].
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