title: Characterization of rabbit aldose reductase-like protein with 3β-hydroxysteroid dehydrogenase activity.
abstract: In this study, we isolated the cDNA for a rabbit aldose reductase-like protein that shared an 86% sequence identity to human aldo-keto reductase (AKR)(1) 1B10 and has been assigned as AKR1B19 in the AKR superfamily. The purified recombinant AKR1B19 was similar to AKR1B10 and rabbit aldose reductase (AKR1B2) in the substrate specificity for various aldehydes and α-dicarbonyl compounds. In contrast to AKR1B10 and AKR1B2, AKR1B19 efficiently reduced 3-keto-5α/β-dihydro-C19/C21/C24-steroids into the corresponding 3β-hydroxysteroids, showing K(m) of 1.3-9.1 μM and k(cat) of 1.1-7.6 min(-1). The stereospecific reduction was also observed in the metabolism of 5α- and 5β-dihydrotestosterones in AKR1B19-overexpressing cells. The mRNA for AKR1B19 was ubiquitously expressed in rabbit tissues, and the enzyme was co-purified with 3β-hydroxysteroid dehydrogenase activity from the lung. Thus, AKR1B19 may function as a 3-ketoreductase, as well as a defense system against cytotoxic carbonyl compounds in rabbit tissues. The molecular determinants for the unique 3-ketoreductase activity were investigated by replacement of Phe303 and Met304 in AKR1B19 with Gln and Ser, respectively, in AKR1B10. Single and double mutations (F303Q, M304S and F303Q/M304S) significantly impaired this activity, suggesting the two residues play critical roles in recognition of the steroidal substrate.
authors.affiliation: Laboratory of Biochemistry, Gifu Pharmaceutical University, Gifu 501-1196, Japan. sendo@gifu-pu.ac.jp
authors.forename: Satoshi
authors.initials: S
authors.lastname: Endo
authors.forename: Toshiyuki
authors.initials: T
authors.lastname: Matsunaga
authors.forename: Sho
authors.initials: S
authors.lastname: Kumada
authors.forename: Airi
authors.initials: A
authors.lastname: Fujimoto
authors.forename: Satoshi
authors.initials: S
authors.lastname: Ohno
authors.forename: Ossama
authors.initials: O
authors.lastname: El-Kabbani
authors.forename: Dawei
authors.initials: D
authors.lastname: Hu
authors.forename: Naoki
authors.initials: N
authors.lastname: Toyooka
authors.forename: Jun'ichi
authors.initials: J
authors.lastname: Mano
authors.forename: Kazuo
authors.initials: K
authors.lastname: Tajima
authors.forename: Akira
authors.initials: A
authors.lastname: Hara
mesh_terms: D015096:3-Hydroxysteroid Dehydrogenases / Q000378:metabolism*; D000449:Aldehyde Reductase / Q000032:analysis* / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000447:Aldehydes / Q000378:metabolism*; D000074408:Aldo-Keto Reductases; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D016297:Mutagenesis, Site-Directed; D010084:Oxidation-Reduction; D011817:Rabbits / Q000235:genetics / Q000378:metabolism*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013256:Steroids / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: C403894:4-oxo-2-nonenal; D000447:Aldehydes; D018076:DNA, Complementary; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D013256:Steroids; D015096:3-Hydroxysteroid Dehydrogenases; C120471:AKR1B10 protein, human; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase