Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by tricarboxylic acid cycle metabolites

In Bacillus subtilis, recent in vivo studies revealed that particular enzymes of the tricarboxylic acid cycle form complexes that allow an efficient transfer of metabolites. Remarkably, a complex of the malate dehydrogenase (Mdh) (EC 1.1.1.37) with isocitrate dehydrogenase (Icd) (EC 1.1.1.42) was identified, although both enzymes do not catalyze subsequent reactions. In the present study, the interactions between these enzymes were characterized in vitro by surface plasmon resonance in the absence and presence of their substrates and cofactors. These analyses revealed a weak but specific interaction between Mdh and Icd, which was specifically stimulated by a mixture of substrates and cofactors of Icd: isocitrate, NADP+ and Mg2+. Wild‐type Icd converted these substrates too fast, preventing any valid quantitative analysis of the interaction with Mdh. Therefore, binding of the IcdS104P mutant to Mdh was quantified because the mutation reduced the enzymatic activity by 174‐fold but did not affect the stimulatory effect of substrates and cofactors on Icd–Mdh complex formation. The analysis of the unstimulated Mdh–IcdS104P interaction revealed kinetic constants of ka = 2.0 ± 0.2 × 102 m−1·s−1 and kd = 1.0 ± 0.1 × 10−3·s−1 and a KD value of 5.0 ± 0.1 μm. Addition of isocitrate, NADP+ and Mg2+ stimulated the affinity of IcdS104P to Mdh by 33‐fold (KD = 0.15 ± 0.01 μm, ka = 1.7 ± 0.7 × 103 m−1·s−1, kd = 2.6 ± 0.6 × 10−4·s−1). Analyses of the enzymatic activities of wild‐type Icd and Mdh showed that Icd activity doubles in the presence of Mdh, whereas Mdh activity was slightly reduced by Icd. In summary, these data indicate substrate control of complex formation in the tricarboxylic acid cycle metabolon assembly and maintenance of the α‐ketoglutarate supply for amino acid anabolism in vivo. Structured digital Idh physically interacts with Mdh by cross-linking study (View interaction) Mdh binds to Idh by surface plasmon resonance (1, 2) The study revealed a strong influence of isocitrate, NADP+ and Mg2+ on the interaction of the TCA cycle enzymes malate dehydrogenase and isocitrate hydrogenase in surface plasmon resonance analyses and an exclusive stimulation of isocitrate dehydrogenase activity by Mdh in enzyme activity assays. These data suggest substrate control of TCA cycle metabolon assembly and maintenance of α‐ketoglutarate supply in vivo.