ABSTRACT
NÉ-lysine acetylation is one of the most abundant post-translational modifications in eukaryote and prokaryote. Protein acetylome of Escherichia coli has been screened using mass spectrometry (MS) technology, and many acetylated proteins have been identified, including the pyridoxine 5'-phosphate oxidase (EcPNPOx), but the biological roles played by lysine acetylation in EcPNPOx still remain unknown. In this study, EcPNPOx was firstly overexpressed and purified, and two acetylated lysine residues were identified by the subsequent liquid chromatography-tandem mass spectrometry analysis. Site-directed mutagenesis analysis demonstrated that acetylated lysine residues play important roles in the enzymatic activity and enzymatic properties of the protein. EcPNPOx could be non-enzymatically acetylated by acetyl-phosphate and deacetylated by CobB in vitro. Furthermore, enzymatic activities of acetylated and deacetylated EcPNPOx were compared in vitro, and results showed that acetylation led to a decrease of its enzymatic activity, which could be rescued by CobB deacetylation. Taken together, our data suggest that CobB modulates the enzymatic activity of EcPNPOx in vitro.
Subject(s)
Escherichia coli Proteins/metabolism , Escherichia coli/enzymology , Lysine/metabolism , Protein Processing, Post-Translational , Pyridoxaminephosphate Oxidase/metabolism , Acetylation , Amino Acid Sequence , Blotting, Western , Chromatography, Liquid , Enzyme Assays/methods , Enzyme Stability , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/genetics , Hydrogen-Ion Concentration , Lysine/genetics , Mutagenesis, Site-Directed , Pyridoxaminephosphate Oxidase/genetics , Sequence Homology, Amino Acid , Sirtuins/metabolism , Tandem Mass Spectrometry , TemperatureABSTRACT
Lysine acetylation is one of the most abundant post-translational modifications. However, physiological roles of this modification in bacteria are largely unknown. Previous protein acetylome analysis showed that Escherichia coli adenosylmethionine synthase (MAT) undergoes acetylation in vivo, but the biological functions of this modification still need to be uncovered. In this study, MAT of E. coli was over-expressed and purified. Subsequent mass spectrometry analysis showed that 12 lysine residues of the protein were acetylated. Site-directed mutagenesis analysis was performed and the results showed that acetylated lysine residues play important roles in the enzymatic activity of MAT. Next, deacetylation assay was performed by using CobB as the deacetylase, and the results showed that CobB could deacetylate MAT in vitro In addition, the enzymatic activities of acetylated and deacetylated MAT were compared in vitro, and results showed that acetylation led to a decrease in its enzymatic activity, which could be reversed by CobB deacetylation. Altogether, our data suggest that CobB modulates the enzymatic activity of E. coli MAT in vitro.
