Non-enzymatic acetylation inhibits glycolytic enzymes in Escherichia coli.

Advanced mass spectrometry methods have detected thousands of post-translational phosphorylation and acetylation sites in bacteria, but their functional role and the enzymes catalyzing these modifications remain largely unknown. In addition to enzymatic acetylation, lysine residues can also be chemically acetylated by the metabolite acetyl phosphate. In Escherichia coli, acetylation at over 3,000 sites has been linked to acetyl phosphate, but the functionality of this widespread non-enzymatic acetylation is even less clear than the enzyme-catalyzed one. Here, we investigate the role of acetyl-phosphate-mediated acetylation in E. coli central metabolism. Out of 19 enzymes investigated, only GapA and GpmA are acetylated at high stoichiometry, which inhibits their activity by interfering with substrate binding, effectively reducing glycolysis when flux to or from acetate is high. Extrapolating our results to the whole proteome, maximally 10% of the reported non-enzymatically acetylated proteins are expected to reach a stoichiometry that could inhibit their activity.

Extensive regulation of enzyme activity by phosphorylation in Escherichia coli.

Protein serine/threonine/tyrosine (S/T/Y) phosphorylation is an essential and frequent post-translational modification in eukaryotes, but historically has been considered less prevalent in bacteria because fewer proteins were found to be phosphorylated and most proteins were modified to a lower degree. Recent proteomics studies greatly expanded the phosphoproteome of Escherichia coli to more than 2000 phosphorylation sites (phosphosites), yet mechanisms of action were proposed for only six phosphosites and fitness effects were described for 38 phosphosites upon perturbation. By systematically characterizing functional relevance of S/T/Y phosphorylation in E. coli metabolism, we found 44 of the 52 mutated phosphosites to be functional based on growth phenotypes and intracellular metabolome profiles. By effectively doubling the number of known functional phosphosites, we provide evidence that protein phosphorylation is a major regulation process in bacterial metabolism. Combining in vitro and in vivo experiments, we demonstrate how single phosphosites modulate enzymatic activity and regulate metabolic fluxes in glycolysis, methylglyoxal bypass, acetate metabolism and the split between pentose phosphate and Entner-Doudoroff pathways through mechanisms that include shielding the substrate binding site, limiting structural dynamics, and disrupting interactions relevant for activity in vivo.