Substrate specificities and functional properties of human short-chain dehydrogenases/reductases

Abstract: Short-chain dehydrogenases/reductases (SDRs) constitute a universal superfamily of functionally heterogeneous proteins and participate in the metabolism of steroids, prostaglandins, retinoids, aliphatic alcohols, and xenobiotics. So far, more than 3000 members including species variants and over 60 human SDR genes are deposited in databases. Several human SDR genes are involved in different steroid-dependent cancer forms and metabolic disorders by virtue of their hormone metabolizing properties, which make these SDR enzymes interesting novel targets for drug development. The main focus of the thesis is on three human SDR enzymes, type 1 11beta-hydroxy steroid dehydrogenase (11beta-HSD1), type 10 17beta-hydroxysteroid dehydrogenase (17beta-HSDIO) and the functionally unannotated protein Hep27. 11beta-HSD1 was studied by analyzing its enzymological properties, substrate specificities, structure-function relationships and inhibition profiles using different mammalian orthologs. The kinetic comparison of 11beta-HSD1 from the glucocorticoid resistant guinea pig versus glucocorticoid sensitive human species shows that both isoforms display Michaelis-Menten kinetics and similar kinetic constants, indicating that I 11beta-HSD1 is not a critical determinant of peripheral glucocorticoid resistance in guinea pig. A novel role of rodent and human 11betaHSD1 in oxysterol metabolism as a 7oxo-reductase and 7beta-dehydrogenase was discovered. This novel finding indicates a possible involvement of human 11beta-HSD1 in atherosclerotic lesion formation and links glucocorticoid and cholesterol metabolism. Furthermore, the active site variability in 11beta-HSD1 species was investigated by performing inhibition studies, using non-selective steroid-like and selective arylsulfonamidothiazole compounds, and by determining the primary structures of 11beta-HSD1 variants from several species. The results reveal significant differences within the active site architecture of 110-HSDI isozymes, and provide information helpful for further inhibitor design. To understand the physiological role of the multifunctional enzyme 17beta-HSD1O, substrate specificities of human and Drosophila 17beta-HSD10 were investigated. Both orthologs display similar affinities towards estrogen, androgen and hydroxyacyl-CoAs. Whereas human 17betaHSD10 catalyzes conversion of 7alpha-OH and 7beta-OH bile acids, the Drosophila enzyme converts only 7alpha-OH steroids. hi addition, 20P-OH and 21-OH activities of C21 steroids were detected for both orthologs. Homology modeling of the enzyme variants, based on the highresolution crystal structure of rat 17beta-HSD10, and substrate docking reveal a large hydrophobic substrate pocket, able to accommodate steroids of different configurations. The mitochondrial targeting of human 17beta-HSD10 was studied by using hybrid constructs with green fluorescent protein. Residues 1-34 at the N-terminal were shown to consist of a noncleavable mitochondrial target sequence. Residues 1-15 are specific for the enzyme but not sufficient for protein import. Residues 16-34 can be replaced by similar SDR structures such as the corresponding sequence in 3beta/17beta-HSD. On the other hand Drosophila 170-HSD10 shows a cytosolic localization pattern, due to an N-terminal sequence difference. Cross-species comparisons revealed that Hep27 is member of a highly conserved SDR cluster found in human, C. elegans, Drosophila and A. thaliana In this thesis a substrate screening was performed using a compound library, comprising a small set of steroids, retinoids, sugars and several miscellaneous xenobiotic carbonyl compounds. The results show that Hep27 is an NADPH-dependent dicarbonyl reductase, catalyzing the conversion of 3,4-hexanedione, 2,3heptanedione and 1-phenyl-1,2-propanedione. Expression analysis reveals that Hep27 is expressed in human endothelial tissues. These results suggest that Hep27 constitutes a detoxification mechanism within the endothelium against reactive alpha-dicarbonyls.