title: Identification of a determinant for strict NADP(H)-specificity and high sensitivity to mixed-type steroid inhibitor of rabbit aldo-keto reductase 1C33 by site-directed mutagenesis.
abstract: In rabbit tissues, hydroxysteroid dehydrogenase belonging to the aldo-keto reductase (AKR) superfamily exists in six isoforms (AKRs: 1C5 and 1C29-1C33), sharing >73% amino acid sequence identity. AKR1C33 is strictly NADPH-specific, in contrast to dual NADPH/NADH specificity of the other isoforms. All coenzyme-binding residues of the structurally elucidated AKR1C5 are conserved in other isoforms, except that S217 (interacting with the pyrophosphate moiety) and T273 (interacting with the 2'-phosphate moiety) are replaced with F217 and N272, respectively, in AKR1C33. To explore the determinants for the NADPH specificity of AKR1C33, we prepared its F217S and N272T mutant enzymes. The mutation of F217S, but not N272T, converted AKR1C33 into a dually coenzyme-specific form that showed similar kcat values for NAD(P)H to those of AKR1C32. The reverse mutation (S217F) in dually coenzyme-specific AKR1C32 produced a strictly NADPH-specific form. The F217S mutation also abolished the activity towards 3-keto-5β-cholestanes that are substrates specific to AKR1C33, and markedly decreased the sensitivity to 4-pregnenes (such as deoxycorticosterone and medroxyprogesterone acetate) that were found to be potent mixed-type inhibitors of the wild-type enzyme. The results indicate the important role of F217 in the strict NADPH-dependency, as well as its involvement in the unique catalytic properties of AKR1C33.
authors: Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu, Japan. Electronic address: sendo@gifu-pu.ac.jp.
authors: Satoshi
authors: S
authors: Endo
authors: Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu, Japan.
authors: Toshiyuki
authors: T
authors: Matsunaga
authors: Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu, Japan.
authors: Akira
authors: A
authors: Ikari
authors: Monash Institute of Pharmaceutical Sciences, Monash University, Victoria, Australia.
authors: Ossama
authors: O
authors: El-Kabbani
authors: Faculty of Engineering, Gifu University, Gifu, Japan.
authors: Akira
authors: A
authors: Hara
authors: Faculty of Engineering, Gifu University, Gifu, Japan.
authors: Yukio
authors: Y
authors: Kitade
mesh_terms: D000449:Aldehyde Reductase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D000074408:Aldo-Keto Reductases; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D020134:Catalytic Domain / Q000235:genetics; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000494:pharmacology; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D011817:Rabbits; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013256:Steroids / Q000737:chemistry / Q000494:pharmacology; D013379:Substrate Specificity
chemical_list: D004791:Enzyme Inhibitors; D007527:Isoenzymes; D011994:Recombinant Proteins; D013256:Steroids; D009249:NADP; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase
keywords: Aldo–keto reductase; Bile acid synthesis; Coenzyme specificity determinant; Conformational change; Deoxycorticosterone; Hydroxysteroid dehydrogenase