Non-consecutive enzyme interactions within TCA cycle supramolecular assembly regulate carbon-nitrogen metabolism

Area of Science:

• Biochemistry
• Metabolic Engineering
• Systems Biology

Background:

• Enzymes in central metabolism form transient supramolecular complexes.
• The functional role of interactions between non-consecutive metabolic enzymes is not well understood.

Purpose of the Study:

• To investigate the functional significance of interactions between malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD) from Bacillus subtilis.
• To elucidate the impact of MDH-ICD interactions on enzyme activity and metabolic fluxes.

Main Methods:

• Co-localization of MDH and ICD in phase-separated droplets.
• In vitro enzyme activity assays.
• Theoretical modeling of enzyme clustering.
• In vivo metabolic flux analysis in Bacillus subtilis.

Main Results:

• MDH-ICD interaction induced enzyme agglomeration, enhancing ICD catalytic rate.
• 2-oxoglutarate, ICD's product, was sequestered upon MDH-ICD interaction.
• MDH overexpression in vivo led to 2-oxoglutarate accumulation, reduced carbon-nitrogen metabolism fluxes, impaired ammonium assimilation, and decreased biomass.
• Theoretical modeling supported MDH-mediated ICD clustering as the cause of observed phenomena.

Conclusions:

• The interaction between MDH and ICD is crucial for coordinating carbon-nitrogen metabolism in Bacillus subtilis.
• Enzyme complex formation can regulate metabolic pathways and cellular growth.