A mixer tap mechanism in Mycobacterium tuberculosis regulates a critical metabolic node in response to nutrient availability

Mycobacterium tuberculosis persists within the host by exploiting metabolic flexibility underpinned by the tricarboxylic acid (TCA) cycle-glyoxylate shunt junction, where isocitrate dehydrogenase (ICD) and isocitrate lyase (ICL) compete to direct carbon flux between energy production and carbon conservation. Yet, how nutrient availability regulates flux partitioning at this junction is unknown. We show that among the four gatekeeping isoforms of isocitrate lyase (ICL1/2) and isocitrate dehydrogenase (ICD1/2), only icl1 transcription substantially responds to carbon substrate switching. Systematic metabolite screening revealed that pyruvate, oxaloacetate, and glyoxylate are novel allosteric activators of ICD2, while concurrently inhibiting ICL1/2 at their physiological concentrations. To resolve how these regulatory layers integrate, we developed an experimental ¹ H NMR-based flux assay that combines recombinant enzymes with metabolomics-informed metabolite conditions to quantify flux partitioning under defined states. This systems-level framework reveals that flux partitioning emerges from coordinated balancing of enzyme abundance and protein-metabolite effector combinations that shift in a time-dependent, nutrient-responsive manner. This dynamic, multi-layered regulatory framework, analogous to a ‘mixer-tap’ mechanism, offers new strategies for therapeutic disruption of M. tuberculosis metabolism.