title: Structure and quantum chemical analysis of NAD+-dependent isocitrate dehydrogenase: hydride transfer and co-factor specificity.
abstract: The crystal structure of Acidithiobacillus thiooxidans isocitrate dehydrogenase complexed with NAD+ and citrate has been solved to a resolution of 1.9 A. 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 degrees . 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.
authors.affiliation: Department of Frontier Biosciences, Osaka University, 1-3 Yamadaoka, Suita, Osaka 565-0871, Japan. kimada@fbs.osaka-u.ac.jp
authors.forename: Katsumi
authors.initials: K
authors.lastname: Imada
authors.forename: Takashi
authors.initials: T
authors.lastname: Tamura
authors.forename: Ryo
authors.initials: R
authors.lastname: Takenaka
authors.forename: Issei
authors.initials: I
authors.lastname: Kobayashi
authors.forename: Keiichi
authors.initials: K
authors.lastname: Namba
authors.forename: Kenji
authors.initials: K
authors.lastname: Inagaki
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D006859:Hydrogen / Q000737:chemistry*; D006860:Hydrogen Bonding; D007521:Isocitrate Dehydrogenase / Q000737:chemistry* / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009243:NAD / Q000737:chemistry*; D011487:Protein Conformation; D011789:Quantum Theory; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D009243:NAD; D006859:Hydrogen; D007521:Isocitrate Dehydrogenase