Characterization of hamster NAD+-dependent 3(17)b-hydroxysteroid dehydrogenase belonging to the aldo-keto reductase 1C subfamily

The cDNAs for morphine 6-dehydrogenase (AKR1C34) and its homologous aldo-keto reductase (AKR1C35) were cloned from golden hamster liver, and their enzymatic properties and tissue distribution were com-pared. AKR1C34 and AKR1C35 similarly oxidized various xenobiotic alicyclic alcohols using NAD+, but differed in their substrate specificity for hydroxyster-oids and inhibitor sensitivity. While AKR1C34 showed 3a/17b/20a-hydroxysteroid dehydrogenase activities, AKR1C35 efficiently oxidized various 3b- and 17b-hydroxysteroids, including biologically active 3b-hydroxy-5a/b-dihydro-C19/C21-steroids, dehydroe-piandrosterone and 17b-estradiol. AKR1C35 also dif-fered from AKR1C34 in its high sensitivity to flavonoids, which inhibited competitively with respect to 17b-estradiol (Ki 0.110.69 kM). The mRNA for AKR1C35 was expressed liver-specific in male ham-sters and ubiquitously in female hamsters, whereas the expression of the mRNA for AKR1C34 displayed opposite sexual dimorphism. Because AKR1C35 is the first 3(17)b-hydroxysteroid dehydrogenase in the AKR superfamily, we also investigated the molecular deter-minants for the 3b-hydroxysteroid dehydrogenase activ-ity by replacement of Val54 and Cys310 in AKR1C35 with the corresponding residues in AKR1C34, Ala and Phe, respectively. The mutation of Val54Ala, but not Cys310Phe, significantly impaired this activity, sug-gesting that Val54 plays a critical role in recognition of the steroidal substrate