Charlotta Filling

Forms and functions of human SDR enzymes

Short-chain dehydrogenases/reductases (SDR) are defined by distinct, common sequence motifs but constitute a functionally heterogenous superfamily of enzymes. At present, well over 1600 members from all forms of life are annotated in databases. Using the defined sequence motifs as queries, 37 distinct human members of the SDR family can be retrieved. The functional assignments of these forms fall minimally into three main groups, enzymes involved in intermediary metabolism, enzymes participating in lipid hormone and mediator metabolism, and open reading frames (ORFs) of yet undeciphered function. This overview, prepared just before completion of the human genome project, gives the different human SDR forms and relates them to human diseases.

Expanded substrate screenings of human and Drosophila type 10 17beta-hydroxysteroid dehydrogenases (HSDs) reveal multiple specificities in bile acid and steroid hormone metabolism: characterization of multifunctional 3alpha/7alpha/7beta/17beta/20beta/21-HSD.

17beta-hydroxysteroid dehydrogenases (17beta-HSDs) catalyse the conversion of 17beta-OH (-hydroxy)/17-oxo groups of steroids, and are essential in mammalian hormone physiology. At present, eleven 17beta-HSD isoforms have been defined in mammals, with different tissue-expression and substrate-conversion patterns. We analysed 17beta-HSD type 10 (17beta-HSD10) from humans and Drosophila, the latter known to be essential in development. In addition to the known hydroxyacyl-CoA dehydrogenase, and 3alpha-OH and 17beta-OH activities with sex steroids, we here demonstrate novel activities of 17beta-HSD10. Both species variants oxidize the 20beta-OH and 21-OH groups in C21 steroids, and act as 7beta-OH dehydrogenases of ursodeoxycholic or isoursodeoxycholic acid (also known as 7beta-hydroxylithocholic acid or 7beta-hydroxyisolithocholic acid respectively). Additionally, the human orthologue oxidizes the 7alpha-OH of chenodeoxycholic acid (5beta-cholanic acid, 3alpha,7alpha-diol) and cholic acid (5beta-cholanic acid). These novel substrate specificities are explained by homology models based on the orthologous rat crystal structure, showing a wide hydrophobic cleft, capable of accommodating steroids in different orientations. These properties suggest that the human enzyme is involved in glucocorticoid and gestagen catabolism, and participates in bile acid isomerization. Confocal microscopy and electron microscopy studies reveal that the human form is localized to mitochondria, whereas Drosophila 17beta-HSD10 shows a cytosolic localization pattern, possibly due to an N-terminal sequence difference that in human 17beta-HSD10 constitutes a mitochondrial targeting signal, extending into the Rossmann-fold motif.

Structure-Function Relationships of SDR Hydroxysteroid Dehydrogenases

Since its discovery and subsequent establishment as a superfamily, rapid progress on the knowledge of short chain dehydrogenases/reductases (SDR) has been achieved (Jornvall et al., 1981; Persson et al., 1991; Jornvall et al., 1995). Based on conserved sequence characteristics, over 100 different enzymes in the databases now belong to the SDR family. The conserved residues are restricted to certain sequence segments and include the coenzyme binding site and the catalytic center (Krook et al., 1990; Persson et al., 1991; Krozowski, 1994; Ghosh et al., 1994; Jornvall et al., 1995). Despite a residue identity level of 20–30% between different SDR members, the 3D structures thus far analyzed (Ghosh et al., 1991, 1994; Varughese et al., 1992; Ghosh et al., 1995; Tanaka et al., 1996a,b; Benach et al., 1996) reveal a highly similar architecture with a one-domain α/β folding pattern. In particular, most of the conserved residues are found at positions 10–40 (comprising strands βA and βB, helix αB and the joining turns), 80–90 (strand βD), 110 (in helix αE), 130–180 (strands βE and βF, and helix αF) and 183–184 (at the end of strand βF) in the 3β/17β-hydroxysteroid dehydrogenase numbering system. Table 1 lists these segments with residues conserved in more than 80% of all SDR structures and relates them to the secondary structure elements. Sequence comparisons, chemical modifications, site-directed mutageneses and crystallographic analyses reveal that most of these parts form the coenzyme binding and catalytic sites of SDR proteins, thus establishing the secondary structure elements βA to αD as parts of the coenzyme binding site with a typical “Rossmann fold” and a highly conserved Y-X-X-X-K segment (residues 150–154, helix αF) as part of the catalytic center (Persson et al., 1991; Ghosh et al., 1994; Jornvall et al., 1995).

Subcellular targeting analysis of SDR-type hydroxysteroid dehydrogenases.

Most mammalian hydroxysteroid dehydrogenases known thus far belong to the protein superfamilies of short-chain dehydrogenases/reductases (SDR) and aldo-keto reductases (AKR). Whereas members of the AKR family are soluble, cytoplasmic enzymes, SDR-type hydroxysteroid dehydrogenases are also located to other subcellular compartments, i.e. endoplasmic reticulum, mitochondria or peroxisomes. Differential localization might play an important role in influencing the reaction direction of hydroxy dehydrogenase/oxo reductase pathways by determining the available nucleotide cofactor pool. Targeting signals for different subcellular organelles in human hydroxysteroid dehydrogenases have been identified, however, in several enzymes localization signals remain to be determined.

Lack of quinone reductase activity suggests that amyloid-beta peptide/ERAB induced lipid peroxidation is not directly related to production of reactive oxygen species by redoxcycling.

Mitochondrial type II hydroxyacyl-CoA dehydrogenase (ERAB) has recently been shown to mediate amyloid-beta peptide (Abeta) induced apoptosis and neurodegeneration. The precise mechanism of cell death induction is unknown, however, Abeta inhibits ERAB activities and as a result of ERAB-Abeta interactions, enhanced formation of lipid peroxidation products occur. The possibility that ERAB mediates quinone reduction is therefore investigated, thus giving the potential of redoxcycling and production of reactive oxygen species, leading to lipid peroxidation. Recombinant human ERAB was produced in a bacterial expression system and enzymological properties were evaluated. Using several orthoquinones as substrates, no ERAB mediated quinone reductase activity was found either in the presence or absence of Abeta, suggesting that the observed in vivo lipid peroxidation is a result of other mechanisms than redoxcycling by quinones.

Structure-function relationships of 3 beta-hydroxysteroid dehydrogenases involved in bile acid metabolism.

In vertebrates, 3 β-hydroxysteroid dehydrogenases (3β-HSDs) fulfill several physiological and metabolic functions. They are primarily involved in the synthesis of all classes of steroid hormones by catalyzing the 3β-OH dehydrogenation/Δ4–5 isomerization of steroid hormone precursor molecules (Simard et al., 1996). In mammals they furthermore mediat3-hydroxyl group epimerization of bile acids and other steroids during en-terohepatic circulation (Figure 1). The enzymes involved in this reaction comprise 3σ-x-hydroxysteroid dehydrogenases (3a-HSD), 3-keto reductases and 3β-HSDs. Epimerization of steroids secreted in the bile occurs in the intestine, catalyzed by microbial hydroxysteroid dehydrogenases and in the liver. The importance of these epimerization reactions is unknown, but it is anticipated that liver 3β-HSDs are involved in intracellular bindinnd transport of bile acids from the sinusoidal to the canalicular side of the hepatocyte thus might play a role in cholestatic processes (Marschall et al., 1998).