Cloning, expression and site-directed mutagenesis of the rat liver 3-alpha-hydroxysteroid dehydrogenase
Abstract
Hydroxysteroid dehydrogenases (HSDs) are essential enzymes for the biosynthesis and degradation of steroid hormones. Metabolites of the 3$\alpha$-HSD pathway are implicated in the development of prostatic hypertrophy and 3$\alpha$-hydroxy-metabolites of progesterone in the CNS have sedative hypnotic properties due to their interaction with the GABA receptor. The highest levels of 3$\alpha$-HSD expression are seen in rat liver and this enzyme has been well characterized. Rat liver 3$\alpha$-HSD has been purified in this laboratory and in addition to catalyzing the oxidoreduction of glucocorticoids, androgens and progestins, it also catalyzes the oxidation of trans-dihydrodiols of polycyclic aromatic hydrocarbons to reactive ortho-quinones and thereby may represent a pathway of carcinogen activation. Hepatic 3$\alpha$-HSD is also involved in bile acid metabolism and can be potently inhibited by nonsteroidal anti-inflammatory drugs. The ability of 3$\alpha$-HSD to recognize these diverse substrates made elucidation of its structure a primary goal of the laboratory. The structural determination of 3$\alpha$-HSD involved affinity-labeling, x-ray crystallography, and the molecular biological approaches of cloning and site-directed mutagenesis. Prior to the beginning of this project, no HSDs had been cloned and little was known about the structural relationships between 3$\alpha$-HSD and other HSDs. Obtaining the sequence for rat liver 3$\alpha$-HSD demonstrated that it was not structurally similar with other HSDs including human placental 17$\beta$-HSD and rat liver 11$\beta$-HSD, except that it did contain a conserved Tyr-X-X-X-Lys pentapeptide seen in other HSDs. This tyrosine was subsequently found to be essential for catalysis in 11$\beta$-HSD. The greatest degree of sequence identity was seen between 3$\alpha$-HSD and aldose reductase and other members of the aldo-keto reductase family. The x-ray crystal structure of human placental aldose reductase has recently been determined, and using it as a search model in conjunction with the deduced amino acid from the cDNA, the x-ray crystal structure of rat liver 3$\alpha$-HSD has been solved. The crystal structures of aldose reductase and 3$\alpha$-HSD suggested that Tyr-55 could act as the general acid of catalysis. Mutagenesis of Tyr-55 to Phe-55 abolished enzyme activity. In contrast, mutating the tyrosine residue which was conserved with the other HSDs had no effect on catalysis by 3$\alpha$-HSD. In conjunction with the x-ray crystallographic data, site-directed mutagenesis suggests that Tyr-55 may be the general acid of catalysis in 3$\alpha$-HSD.
Subject Area
Pharmacology
Recommended Citation
Pawlowski, John E, "Cloning, expression and site-directed mutagenesis of the rat liver 3-alpha-hydroxysteroid dehydrogenase" (1993). Dissertations available from ProQuest. AAI9413888.
https://repository.upenn.edu/dissertations/AAI9413888