title: Complex formation between malate dehydrogenase and isocitrate dehydrogenase from Bacillus subtilis is regulated by tricarboxylic acid cycle metabolites.
abstract: 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 Mg(2+). 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 k(a) = 2.0 ± 0.2 × 10(2) m(-1) ·s(-1) and k(d) = 1.0 ± 0.1 × 10(-3) ·s(-1) and a K(D) value of 5.0 ± 0.1 μm. Addition of isocitrate, NADP(+) and Mg(2+) stimulated the affinity of IcdS104P to Mdh by 33-fold (K(D) = 0.15 ± 0.01 μm, k(a) = 1.7 ± 0.7 × 10(3) m(-1) ·s(-1), k(d) = 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.
authors: Lehrstuhl für Mikrobiologie, Department Biologie, Friedrich-Alexander Universität Erlangen-Nürnberg, Germany.
authors: Maike
authors: M
authors: Bartholomae
authors: Frederik M
authors: FM
authors: Meyer
authors: Fabian M
authors: FM
authors: Commichau
authors: Andreas
authors: A
authors: Burkovski
authors: Wolfgang
authors: W
authors: Hillen
authors: Gerald
authors: G
authors: Seidel
mesh_terms: D001412:Bacillus subtilis / Q000201:enzymology*; D002952:Citric Acid Cycle / Q000502:physiology*; D007521:Isocitrate Dehydrogenase / Q000378:metabolism*; D008291:Malate Dehydrogenase / Q000378:metabolism*; D020349:Surface Plasmon Resonance
chemical_list: D008291:Malate Dehydrogenase; D007521:Isocitrate Dehydrogenase
keywords: SPINE; TCA cycle; isocitrate dehydrogenase; malate dehydrogenase; surface plasmon resonance