Discussion

Isocitrate dehydrogenase (IDH) is an ubiquitous enzyme catalyzing the decarboxylation of isocitrate to a-ketoglutarate (a-KG) with reduction of a coenzyme. Depending on the types of coenzymes, IDHs are classified as NAD⁺ or NADP⁺-specific IDHs. The parasite Trypanosoma brucei lacks NAD-IDH in its genome, while two NADP-IDHs are localized in mitochondria and glycosomes. The latter (TbIDHg) is essential for differentiation within the insect stage (see abstract Ziebart et al., this meeting), linking glycosomal metabolism and parasite differentiation. In order to obtain insights into the function of TbIDHg, we have purified recombinant TbIDHg and characterized its biochemical properties. Different from other known IDHs, we provided the first evidence that TbIDHg is capable to reduce both NADP⁺ and NAD⁺ at similar catalytic efficiency. Surface Plasmon Resonance analysis confirmed the direct binding of NADPH and NADH with TbIDHg, showed high dissociation constant (K_D) consistent with low affinity and high catalytic efficiency towards both coenzymes. Furthermore, crystal structures of TbIDHg revealed a completely closed state of the active site for the ternary complexes with NAD(P)H/Ca²⁺/a-KG. Interestingly, TbIDHg does not induce a half-closed state of the binary complex with NAD(P)⁺/Ca²⁺, but a completely open state was observed for the first time among all structures of the IDH family, indicating a distinct reaction mechanism from those proposed for other NADP-IDHs.