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.

Conservation of metabolic regulation by phosphorylation and non-covalent small-molecule interactions.

Here, we review extant observations of protein phosphorylation and small-molecule interactions in metabolism and ask which of their specific regulatory functions are conserved in Escherichia coli and Homo sapiens. While the number of phosphosites is dramatically higher in humans, the number of metabolite-protein interactions remains largely constant. Moreover, we found the regulatory logic of metabolite-protein interactions, and in many cases also the effector molecules, to be conserved. Post-translational regulation through phosphorylation does not appear to replace this regulation in human but rather seems to add additional opportunities for fine-tuning and more complex responses. The abundance of metabolite-protein interactions in metabolism, their conserved cross-species abundance, and the apparent conservation of regulatory logic across enormous phylogenetic distance demonstrate their relevance for maintaining cellular homeostasis in these ancient biological processes.