PT - JOURNAL ARTICLE AU - Jasinska, Weronika AU - Dindo, Mirco AU - Correa, Sandra M. AU - Serohijos, Adrian W.R. AU - Laurino, Paola AU - Brotman, Yariv AU - Bershtein, Shimon TI - Non-consecutive enzyme interactions within TCA cycle supramolecular assembly regulate carbon-nitrogen metabolism AID - 10.1101/2023.11.01.565101 DP - 2023 Jan 01 TA - bioRxiv PG - 2023.11.01.565101 4099 - http://biorxiv.org/content/early/2023/11/01/2023.11.01.565101.short 4100 - http://biorxiv.org/content/early/2023/11/01/2023.11.01.565101.full AB - Enzymes of the core energy metabolism pathways tend to assemble into transient supramolecular complexes, yet the functional significance of the interactions within these complexes, particularly between enzymes catalyzing non-consecutive reactions, remains unclear. Here, by co-localizing two non-consecutive enzymes of the TCA cycle from B. subtilis, malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICD), in highly crowded liquid-liquid phase separated droplets we discovered that MDH-ICD interaction causes an enhancement of ICD catalytic rate and an apparent sequestration of its reaction product, 2-oxoglutarate. Theory suggests that the observed phenomena are explained by the MDH-mediating clustering of ICD molecules. In vivo validation with targeted GC-MS and 13C tracer analyses revealed that when B. subtilis is grown on glucose and ammonia, overexpression of MDH leads to accumulation of 2-oxoglutarate with a concomitant reduction of fluxes flowing through both the catabolic and anabolic branches of the carbon-nitrogen intersection occupied by 2-oxoglutarate, resulting in impeded ammonium assimilation and reduced biomass production. Our findings thus suggest that in B. subtilis the MDH-ICD interaction is an important coordinator of carbon-nitrogen metabolism, thereby expanding the list of types of functionally understood unconventional enzyme-enzyme interactions.Competing Interest StatementThe authors have declared no competing interest.