Enzyme–enzyme interactions add another layer of regulation to metabolic pathways in the model organism Bacillus subtilis

Enzymes tend to assemble into supramolecular complexes in response to metabolic cues. However, the functional role of such complexes remains obscure. To address this question, we overexpressed malate dehydrogenase (Mdh) –a TCA-cycle enzyme known to form multiple interactions within the TCA-cycle supramolecular complex in Bacillus subtilis –and measured the ensuing effects on the intracellular metabolite pools using targeted gas-chromatography and mass-spectrometry analysis. To separate possible pleiotropic effects of overexpression caused by sequestration of metabolites or by protein expression burden from genuine effects stemming from perturbation of the TCA supramolecular complex, we compared shifts in the B. subtilis metabolome following overexpression of functional and inactive endogenous Mdh, as well as functional/inactive orthologous Mdhs from closely related species. The rationale behind such an experimental design is rooted in the idea that functional interactions between proteins are expected to be accompanied by mutational co-evolution that preserves the functionality of the interaction. Thus, it is anticipated that orthologous Mdh enzymes that did not take part in the coevolutionary process within B. subtilis will not participate in functional interactions with the TCA-cycle supramolecular complex. Strikingly, when B. subtilis was grown on glucose and ammonia, overexpression of both active and inactive endogenous Mdh resulted in significant shifts in metabolic fluxes flowing through two strategic junctions that control carbon and nitrogen metabolism:oxaloacetate–phosphoenolpyruvate–pyruvate and α-ketoglutarate–glutamate–glutamine. No such effects could be observed with overexpression of active/inactive pairs of orthologous Mdhs, even though their catalytic parameters and expression levels were very similar to those of the endogenous active/inactive Mdh pair.