Transient protein-protein interactions perturbE.colimetabolome and cause gene dosage toxicity

Several genes exhibit gene dosage toxicity yet its molecular underpinnings remain unknown. Here we demonstrate that overexpression of DHFR inE. colicauses toxic metabolic imbalance triggered by interactions with several enzymes involved in 1-carbon metabolism, in particular GlyA and PurH. DHFR overexpression partially inhibits activity of these enzymes, but at physiological concentrations, PurH-DHFR interaction enhances catalytic efficiency of DHFR, implying a functional interactionin vivo. Surprisingly, overexpression of orthologous DHFRs from other bacterial species caused minimal metabolic and fitness perturbations, despite pulling out more interacting partners than overexpressed endogenous DHFR. Orthologous DHFRs were less potent in inhibitingE. coliGlyA and PurH, or gaining a catalytic improvement upon interaction with PurH, indicating a partial loss of interaction specificity due to evolutionary divergence. This study shows how protein overexpression perturbs a dynamic network of weak yet potentially functional PPI with consequences for the metabolic state of cells and their fitness.