Acetylation Regulates The Activity Of Escherichia Coli S-adenosy-L-methionine Synthase

S-adenosyl-L-methionine(Ado Met or SAM) is an important biological sulfonium compound in both prokaryotes and eukaryocytes. According to its capability of donating all the groups surrounding the sulfur atom, SAM is highly reactive and participates in numerous metabolic pathways, like transmethylation reactions, or as a precursor in transsulfuration and aminopropylation. In vivo, SAM can be catalyzed only through the S-adenosylmethionine synthase(MAT, also called methionine adenosyltransferase), encoded by the met K gene, which is well-conserved from bacteria to eukaryotes. Previous protein acetylome analysis showed that, Escherichia coli adenosylmethionine synthase(MAT) undergoes acetylation in vivo, but biological functions of this modification still need to be uncovered.Lysine acetylation of proteins is one of the most prevalent posttranslational modifications(PTMs) in both eukaryotes and prokaryotes. Nowadays, the mechanism of bacterial proteins lysine acetylation can occur by two distinct ways. The conventional mechanism relies on a lysine acetyltransferases(KAT) and transfers the acetyl group from the donation to the ε- amino group of a lysine residue enzymatically. In contrast, the second mechanism is non-enzymatic, which is the predominant mechanism in Escherichia coli. Acetyl-coenzyme A(Ac-Co A) and/or acetyl phosphate(Ac P) directly donates its acetyl group to the lysine ε- amino group.Acetylation can be enzymatically reversed by lysine deacetylases(KDACs). Cob B, as a deacetylase of sirtuin family of NAD+-dependent deacetylases, is best known in bacterium, especially in Escherichia coli. Although lysine acetylation or Cob B mediated deacetylation regulates protein functions in diverse, more biological functions need to be investigated.In this study, MAT of E. coli was first over-expressed and purified. Subsequent mass spectrometry analysis showed that 12 lysine residues were identified to be acetylated. Site directed mutagenesis analysis was performed and the results showed that, acetylated lysine residues play important roles on the enzymatic activity of the protein. Next, deacetylation assay was performed by using Cob B as the deacetylase, and the results showed that Cob B could deacetylate MAT in vitro. In addition, enzymatic activities of acetylated and deacetylated MAT were compared in vitro, and the results showed that acetylation led to a decrease of its enzymatic activity, which could be reversed by Cob B deacetylation. Furthermore, SAM concentration between the wild type strain of W3110 and the cob B gene knockout mutant were compared, the results showed that deletion of cob B led to a significant decrease of SAM concentration inside the bacterial cells. Altogether, our data suggest that Cob B modulates the enzymatic activity of E. coli MAT in vitro. Subsequently, we detected the 5-m C methylation levels of total DNA in E. coli wild-type and Δcob B mutant strain in order to find the relationship between acetylation and DNA methylation. Accidentally, we found MAT could be auto-phosphorylated by using the phosphate donors: acetyl-phosphate and carbamyl phosphate, and could be dephosphorylated by alkaline phosphatase in vitro. The function of MAT phosphorylation needs more investigations.Here we showed that reversible acetylation could regulate the enzymatic activity of MAT in vitro. In addition, intracellular SAM concentration was markedly reduced with the deletion of cob B gene, which encodes the deacetylase in E. coli. These results suggested that Nε-lysine acetylation may affect methionine cycle of E. coli by regulating the activity of MAT.