Short-chain dehydrogenases/reductases : Structure, function and motifs of hydroxysteroid dehydrogenases

Sammanfattning: Short-chain dehydrogenases/reductases (SDR) constitute a superfamily of proteins catalysing reactions of a wide range of substrates in all life forms. During the past years, a large number of new proteins belonging to this superfamily has been detected. They are important in e.g. the metabolism of steroids and bile acids, beta-oxidation and retinol metabolism, and are critical in health and disease. In this thesis, I have focused on hydroxysteroid dehydrogenases in general and three proteins in particular: 3 beta/17 beta-hydroxysteroid dehydrogenase (3 beta/17 beta-HSD) from Comamonas testosteroni, and human and Drosophila 17 beta-hydroxysteroid dehydrogenase type 10/short chain hydroxyacyl coenzyme A dehydrogenase type II (17 beta- HSD10/SCHAD II). 3 beta/17 beta-HSD was studied to obtain information about structure and function relationships and reaction mechanisms within the SDR superfamily. 17 beta-HSD1O/SCHAD II was studied to obtain knowledge about targeting motifs, subcellular organisation of proteins, the functional role in health and disease, and species relationships. 3 beta/17 beta-HSD was studied by site-directed mutagenesis of conserved amino acid residues in the SDR superfamily. Data obtained by steady-state kinetics, stability measurements, X-ray crystallography and docking analysis define roles for several residues regarding different conserved motifs in the cofactor binding site, the catalytic site, and the substrate binding site. The critical role of residues Thr 12, Asp 60, Asn 86, Asti 87, and Ala 88 in coenzyme binding and catalysis is highlighted. The importance of residues regulating the reaction direction, Thr 12 and Asn 87, and the role of Ser 138 involved in the catalytic mechanism, were demonstrated. The data also reveal essential interactions of Asn 111 with the active site residues. A general role of its side-chain interactions in the maintenance of the active site configuration to build tip a proton relay system is proposed. This extends the previously recognized catalytic triad of Set, Tyr, Lys residues to form a tetrad of Asn, Set, Tyr and Lys in the majority of characterised SDR enzymes. The largely conserved residue Asn 179 was concluded to connect the active site and the substrate binding loop, through a conserved water molecule, indicated by loss of function in an N179A mutant in 3 beta/17 beta-HSD. Importantly, this structural motif was detected in 16 out of 21 determined SDR crystal structures. The tertiary and quaternary structures of the apoform of 3 beta/17 beta-HSD have been determined at 1.2 A resolution. The structure, kinetic analysis of wild- type and mutants (Y148F and H182L) with residue exchanges in the binding loop, and docking experiments make it possible to explain the steroid-protein recognition site, with novel structural motifs. The mitochondrial multifunctional enzyme 17 beta-HSD type 10, with activities on 17 beta- and 3 alpha-OH positions on steroids, displays short-chain L-3-hydroxyacyl CoA dehydrogenase activity. This constitutes an essential step in the beta-oxidation of fatty acids, and correspondingly the enzyme is also classified as type 2 short chain hydroxyacyl CoA dehydrogenase (SCHAD II). 17 beta-HSD1O is structurally related to bacterial 3 beta/17 beta-HSD, with about 35% sequence identity. The mitochondrial targeting of the human enzyme was studied by using hybrid constructs with green fluorescent protein. The N-terminal part, residue 1-34, was shown to consist of a non-cleavable mitochondrial target sequence. The first 15 residues are specific for the enzyme but not sufficient to import the functional protein. Residues 16-34 can be replaced by similar structures such as the corresponding sequence in 3 beta/17 beta-HSD. The orthologous, essential protein in Drosophila has been studied in a similar manner and its activities with different steroid substrates, acetoacetyl CoA and DL-beta-hydroxybutyryl CoA, were determined. Material from six patients with defective L-3-hydroxyacyl CoA dehydrogenase activity (the third step in the betaoxidation) for short-chain fatty acids has been studied. Sequences for exons and exon-intron boundaries for SCHAD I and SCHAD II were determined. No mutations in SCHAD II were found, whereas a known SCHAD I polymorphism in two of the six patients was detected. Kinetic comparison of SCHAD I and II reveals that type I is the main enzyme form responsible for beta-oxidation of short- chain hydroxyacyl derivatives of fatty acids in humans. These data suggest that the observed biochemical deficiency leading to the lethal metabolic complications is not a defective SCHAD enzyme, but most likely resides in defective interactions with other proteins, regulating the SCHAD activity.