Structure and quantum chemical analysis of NAD⁺-dependent isocitrate dehydrogenase: Hydride transfer and co-factor specificity

The crystal structure of Acidithiobacillus thiooxidans isocitrate dehydrogenase complexed with NAD⁺ and citrate has been solved to a resolution of 1.9 Å. The protein fold of this NAD⁺-dependent enzyme shares a high similarity with those of NADP⁺-dependent bacterial ICDHs. The NAD⁺ and the citrate are clearly identified in the active site cleft with a well-defined electron density. Asp-357 is the direct cofactor-specificity determinant that interacts with 2′-OH and 3′-OH of the adenosine ribose. The adenosine ribose takes a C2′-endo puckering conformation as previously reported for an NAD⁺-specific isopropylmalate dehydrogenase. The nicotinamide moiety of NAD⁺ has the amide NH₂ group oriented in cis conformation with respect to the C4 carbon of the nicotinamide ring, slanted toward the bound citrate molecule with a dihedral angle of -21°. The semi-empirical molecular orbital calculation suggests that the pro-R hydrogen atom at C4 of NADH would bear the largest negative charge when the amide NH₂ group is in such conformation, suggesting that the amide group has a catalytically significant role in stabilizing the transition state as NADH is being formed during the hydride transfer catalysis.