RT Journal Article SR Electronic T1 Non-consecutive enzyme interactions within TCA cycle supramolecular assembly regulate carbon-nitrogen metabolism JF bioRxiv FD Cold Spring Harbor Laboratory SP 2023.11.01.565101 DO 10.1101/2023.11.01.565101 A1 Jasinska, Weronika A1 Dindo, Mirco A1 Correa, Sandra M. A1 Serohijos, Adrian W.R. A1 Laurino, Paola A1 Brotman, Yariv A1 Bershtein, Shimon YR 2023 UL http://biorxiv.org/content/early/2023/11/01/2023.11.01.565101.abstract 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.