Extensive regulation of enzyme activity by phosphorylation in Escherichia coli

Abstract

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.

Editorial: Post-translational Modification in Response to Stresses in Bacteria

“…The study by Sultan et al in this series supports the recent findings that the bacterial metabolic enzymes are the significant phosphorylation sites by cellular Ser/Thr/Tyr kinases (Schastnaya et al, 2021 ). By employing phosphoproteomic and biochemical approaches, this study has identified impact on the phosphoproteome of Escherichia coli in the absence of hitherto uncharacterized E. coli ser/thr kinase (YeaG).…”

Panoramic Perspective on Human Phosphosites

“…To check whether the above observations are real tendencies for pSTY sites, we replaced every instance of pS by A, pT by V, and pY by F , and predicted the biophysical properties of the modified protein sequence. We observed that the same sequence with modified pSTY > AVF showed an increase in rigidity; all of the values of backbone, disorder, side-chain dynamics, and early fold propensity showed a slight increase in the modified sequence, which illustrates that it is the intrinsic nature of phosphorylatable STY residues to be more dynamic and flexible (Figure S7).…”

A panoramic perspective on human phosphosites

“…To check whether the above observations are real tendencies for pSTY sites, we replaced every instance of pS by A, pT by V and pY by F 70,71 and predicted the biophysical properties for the modified protein sequence. We observed that the same sequence with modified pSTY >AVF showed an increase in rigidity; all the values of backbone, disorder, side chain dynamics and early fold propensity showed a slight increase in the modified sequence, which illustrates that it is the intrinsic nature of phosphorylatable STY residues to be more dynamic and flexible.…”

“…P-sites have lower early folding propensities 60 as compared to non-P-sites, indicating that most of the STY residues involved in so-called foldon (protein region that folds as a unit) formation tend not to be phosphorylated (Figure 11D), which fits with their tendency to be buried. To check whether the above observations are real tendencies for pSTY sites, we replaced every instance of pS by A, pT by V and pY by F 70,71 and predicted the biophysical properties for the modified protein sequence. We observed that the same sequence with modified pSTY>AVF showed an increase in rigidity; all the values of backbone, disorder, side chain dynamics and early fold propensity showed a slight increase in the modified sequence, which illustrates that it is the intrinsic nature of phosphorylatable STY residues to be more dynamic and flexible.…”