Reversible Lysine Acetylation Regulates The Activity Of E.Coli N-Hydroxyarylamine O-Acetyltransferase

Protein lysine acetylation is one of the most widespread protein post-translational modifications in both eukaryotes and bacteria. In recent decades, this post-translational modification has been shown to play key roles in many biological processes in eukaryotic cells. Recently, protein acetylation has also been found to be widespread in bacteria and archaea, however, the extent of this modification in prokaryotic cells remain largely unexplored, thereby presenting a hurdle to further functional study of this modification in prokaryotic systems.Protein acetylation may be controlled by protein acetyltransferases and deacetylases. In bacteria, the best known deacetylase is CobB, which is a homolog of eukaryotic Sir2. Though progress has been made in functional studies of this protein in recent years, its substrates and biological functions are still largely unclear.Using proteome microarray technology, fifteen potential lysine-acetylated proteins and nine potential substrates of Escherichia coli CobB were screened, including N-hydroxyarylamine O-acetyltransferase (NhoA). In vitro acetylation/deacetylation of NhoA was verified by Western blotting and mass spectrometry, and three acetylated lysine residues were identified. Site-specific mutagenesis experiments showed that mutation of two of the acetylated lysines decreased the acetylation level of NhoA in vitro. Further analysis showed that variant NhoA proteins carrying substitutions at these two acetylated lysine residues are involved in both the O-acetyltransferase and N-acetyltransferase activity of NhoA. And then direct data showed that deacetylation of NhoA by CobB increased its NAT activity in vitro. To elucidate the effects of reversible acetylation on NhoA activity in vivo, we tested the acetylation level of NhoA and mutagenicity of nitro aromatic compounds in a cobB-knockout strain. Results showed that, knockout of cobB leads to increased acetylation of NhoA and decreased OAT activity. These results suggest that reversible acetylation regulates the activity of NhoA both in vitro and in vivo, and that CobB could increase the NAT activity in vitro by deacetylating NhoA, it may affect the OAT activity of NhoA in vivo through the same way. Taken together, we identified new lysine acetylated proteins and substrates of deacetylase CobB, and explored a new function of lysine acetylation and bacterial cobB.