Structure of 3-isopropylmalate dehydrogenase in complex with 3-isopropylmalate at 2.0 Å resolution: The role of Glu88 in the unique substrate-recognition mechanism

Background: 3-Isopropylmalate dehydrogenase (IPMDH) and isocitrate dehydrogenase (ICDH) belong to a unique family of bifunctional decarboxylating dehydrogenases. Although the ICDH dimer catalyzes its reaction under a closed conformation, known structures of the IPMDH dimer (without substrate) adopt a fully open or a partially closed form. Considering the similarity in the catalytic mechanism, the IPMDH dimer must be in a fully closed conformation during the reaction. A large conformational change should therefore occur upon substrate binding. Results: We have determined the crystal structure of IPMDH from Thiobacillus ferrooxidans (Tf) complexed with 3-isopropylmalate (IPM) at 2.0 Å resolution by the molecular replacement method. The structure shows a fully closed conformation and the substrate-binding site is quite similar to that of ICDH except for a region around the γ-isopropyl group. The γ group is recognized by a unique hydrophobic pocket, which includes Glu88, Leu91 and Leu92 from subunit 1 and Val193' from subunit 2. Conclusions: A large movement of domain 1 is induced by substrate binding, which results in the formation of the hydrophobic pocket for the γ-isopropyl moiety of IPM. A glutamic acid in domain 1, Glu88, participates in the formation of the hydrophobic pocket. The Cβ and Cγ atoms of Glu88 interact with the γ-isopropyl moiety of IPM and are central to the recognition of substrate. The acidic tip of Glu88 is likely to interact with the nicotinamide mononucleotide (NMN) ribose of NAD⁺ in the ternary complex. This structure clearly explains the substrate specificity of IPMDH.