Reserve Flux Capacity in the Pentose Phosphate Pathway Enables Escherichia coli's Rapid Response to Oxidative Stress

Highlights

• Characterization of the immediate metabolic response of E. coli to oxidative stress

• Development of a high-performance ensemble modeling computational pipeline

• Identification of NADPH feedback inhibition on G6PDH as key regulatory interaction

• Cells without reserve flux capacity in PP pathway are sensitive to oxidative stress

Summary

To counteract oxidative stress and reactive oxygen species (ROS), bacteria evolved various mechanisms, primarily reducing ROS through antioxidant systems that utilize cofactor NADPH. Cells must stabilize NADPH levels by increasing flux through replenishing metabolic pathways like pentose phosphate (PP) pathway. Here, we investigate the mechanism enabling the rapid increase in NADPH supply by exposing Escherichia coli to hydrogen peroxide and quantifying the immediate metabolite dynamics. To systematically infer active regulatory interactions governing this response, we evaluated ensembles of kinetic models of glycolysis and PP pathway, each with different regulation mechanisms. Besides the known inactivation of glyceraldehyde 3-phosphate dehydrogenase by ROS, we reveal the important allosteric inhibition of the first PP pathway enzyme by NADPH. This NADPH feedback inhibition maintains a below maximum-capacity PP pathway flux under non-stress conditions. Relieving this inhibition instantly increases PP pathway flux upon oxidative stress. We demonstrate that reducing cells' capacity to rapidly reroute their flux through the PP pathway increases their oxidative stress sensitivity.