STRUCTURE OF 3-ISOPROPYLMALATE DEHYDROGENASE IN COMPLEX WITH 3-ISOPROPYLMALATE AT 2.0 ANGSTROM RESOLUTION - THE ROLE OF GLU88 IN THE UNIQUESUBSTRATE-RECOGNITION MECHANISM

Background: 3-Isopropylmalate dehydrogenase (IPMDH) and isocitrate deh ydrogenase ([CDH) belong to a unique family of bifunctional decarboxyl ating dehydrogenases. Although the ICDH dimer catalyzes its reaction u nder a closed conformation, known structures of the IPMDH dimer (witho ut substrate) adopt a fully open or a partially closed form. Consideri ng the similarity in the catalytic mechanism, the IPMDH dimer must be in a fully closed conformation during the reaction. A large conformati onal change should therefore occur upon substrate binding. Results: We have determined the crystal structure of IPMDH from Thiobacillus ferr ooxidans (Tf) complexed with 3-isopropylmalate (IPM) at 2.0 Angstrom r esolution by the molecular replacement method. The structure shows a f ully closed conformation and the substrate-binding site is quite simil ar to that of ICDH except for a region around the gamma-isopropyl grou p. The gamma group is recognized by a unique hydrophobic pocket, which includes Glu88, Leu91 and Leu92 from subunit 1 and Val193' from subun it 2. Conclusions: A large movement of domain 1 is induced by substrat e binding, which results in the formation of the hydrophobic pocket fo r the gamma-isopropyl moiety of IPM. A glutamic acid in domain 1, Glu8 8, participates in the formation of the hydrophobic pocket. The C beta and C gamma atoms of Glu88 interact with the gamma-isopropyl moiety o f IPM and are central to the recognition of substrate. The acidic tip of Glu88 is likely to interact with the nicotinamide mononucleotide (N MN) ribose of NAD(+) in the ternary complex. This structure clearly ex plains the substrate specificity of IPMDH.