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.

蛋白质丝氨酸/苏氨酸/酪氨酸 (S/T/Y) 磷酸化是真核生物中一种重要且频繁的翻译后修饰，但历史上一直被认为在细菌中不太普遍，因为发现磷酸化的蛋白质较少，并且大多数蛋白质被修饰为学位较低。最近的蛋白质组学研究将大肠杆菌的磷酸化蛋白质组大大扩展至 2000 多个磷酸化位点（磷酸化位点），但仅提出了 6 个磷酸化位点的作用机制，并描述了 38 个磷酸化位点在扰动时的适应性效应。通过系统地表征大肠杆菌代谢中 S/T/Y 磷酸化的功能相关性，我们根据生长表型和细胞内代谢组谱发现 52 个突变磷酸位点中的 44 个具有功能。通过有效地将已知功能性磷酸位点的数量加倍，我们提供了证据表明蛋白质磷酸化是细菌代谢的主要调节过程。结合体外和体内实验，我们展示了单个磷酸位点如何通过屏蔽底物结合位点、限制酶解、甲基乙二醛旁路、乙酸代谢以及戊糖磷酸和 Entner-Doudoroff 途径之间的分裂来调节酶活性并调节代谢通量。结构动力学，以及破坏与体内活动相关的相互作用。