Erik Nordling

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.

Stabilization of discordant helices in amyloid fibril-forming proteins

Several proteins and peptides that can convert from α‐helical to β‐sheet conformation and form amyloid fibrils, including the amyloid β‐peptide (Aβ) and the prion protein, contain a discordant α‐helix that is composed of residues that strongly favor β‐strand formation. In their native states, 37 of 38 discordant helices are now found to interact with other protein segments or with lipid membranes, but Aβ apparently lacks such interactions. The helical propensity of the Aβ discordant region (K16LVFFAED23) is increased by introducing V18A/F19A/F20A replacements, and this is associated with reduced fibril formation. Addition of the tripeptide KAD or phospho‐L‐serine likewise increases the α‐helical content of Aβ(12–28) and reduces aggregation and fibril formation of Aβ(1–40), Aβ(12–28), Aβ(12–24), and Aβ(14–23). In contrast, tripeptides with all‐neutral, all‐acidic or all‐basic side chains, as well as phosphoethanolamine, phosphocholine, and phosphoglycerol have no significant effects on Aβ secondary structure or fibril formation. These data suggest that in free Aβ, the discordant α‐helix lacks stabilizing interactions (likely as a consequence of proteolytic removal from a membrane‐associated precursor protein) and that stabilization of this helix can reduce fibril formation.

Separate functional features of proinsulin C-peptide

Abstract.Proinsulin C-peptide influences a number of physiological parameters in addition to its well-established role in the parent proinsulin molecule. It is of interest as a candidate for future co-replacement therapy with insulin for patients with diabetes mellitus type 1, but specific receptors have not been identified and additional correlation with functional effects is desirable. Based on comparisons of 22 mammalian proinsulin variants, we have constructed analogues for activity studies, choosing phosphorylation of mitogen-activated protein kinases (MAPKs) in Swiss 3T3 fibroblasts for functional measurements. In this manner, we find that effective phosphorylation of MAPKs is promoted by the presence of conserved glutamic acid residues at positions 3, 11 and 27 of C-peptide and by the presence of helix-promoting residues in the N-terminal segment. Previous findings have ascribed functional roles to the C-terminal pentapeptide segment, and all results combined therefore now show the importance of different segments, suggesting that C-peptide interactions are complex or multiple.

Differential multiplicity of MDR alcohol dehydrogenases: enzyme genes in the human genome versus those in organisms initially studied

Abstract: Screens were made for alcohol dehydrogenase (ADH) of the classical type (the MDR superfamily) in translations of human and other relevant genomes, corresponding to the organism types from which the enzyme was initially purified. Considerable multiplicities were detected in the dimeric enzymes from higher eukaryotes: seven forms in the human (plus three pseudogenes), all genes on chromosome 4, in the order class IV → class Iγ→ class Iβ→ class Iα→ class V → class II → class III, and eight forms in Arabidopsis thaliana (plus one pseudogene). These multiplicity patterns, and the species variability in the animal (human/mouse) and plant (Arabidopsis/pea) lines, suggest parallel but separate duplicatory events, giving rise to three families of dimeric MDR-ADH: class III, the animal non-class III, and the plant non-class III enzymes, with functions in formaldehyde elimination, in alcohol/aldehyde detoxication and in special pathways in higher eukaryotes. Multiplicity, although to a lesser extent, was also noted in tetrameric MDR-ADH, suggesting functional divergence between the dimeric and tetrameric enzymes. Combining these observations, at least five levels of divergence are reflected in the present ADH forms, corresponding to nodes at the SDR/MDR, the dimer/tetramer, the class III/non-class III, the class I/P, and the more recent class splits, each branch associated with separate functional patterns.

Identification and characterisation of two allelic forms of human alcohol dehydrogenase 2

Abstract. The human alcohol dehydrogenase system is comprised of multiple forms that catalyse the oxidation/reduction of a large variety of alcohols and aldehydes. A transition that results in an Ile308Val substitution was identified in the human ADH2 gene by single-strand conformation polymorphism analysis. Screening a Swedish population revealed that Val308 was the most frequent allele (73%), and site-directed mutagenesis was used to obtain both allelozymes, which were expressed in Escherichia coli for characterisation. Thermostability was assayed by activity measurements and circular dichroism spectroscopy. The results showed that the 308Val substitution decreases protein stability, as compared to the Ile308 variant, an effect also demonstrated during prolonged storage. Ethanol, octanol, 12-hydroxydodecanoic acid and all-trans retinol were used as model substrates and, generally, slightly higher Km values were observed with Val at position 308. Finally, homology modelling, from mouse ADH2, further supported the decreased stability of the Val308 variant and located position 308 in the subunit interface of the molecule and in the vicinity of the active-site pocket entrance. In conclusion, the Ile308Val substitution represents a novel functional polymorphism within the human alcohol dehydrogenase gene cluster that may affect the metabolism of ethanol and other substrates.

Human type 10 17 beta-hydroxysteroid dehydrogenase: molecular modelling and substrate docking.

17 beta-hydroxysteroid dehydrogenases catalyze the oxidoreduction of hydroxy/oxo groups at position C17 of steroid hormones, thereby constituting a prereceptor control mechanism of hormone action. At present, 11 different mammalian 17 beta-hydroxysteroid dehydrogenases have been identified, catalyzing the cell- and steroid-specific activation and inactivation of estrogens and androgens. The human type 10 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD-10) is a multifunctional mitochondrial enzyme that efficiently catalyzes the oxidative inactivation at C17 of androgens and estrogens. However, it also mediates oxidation of 3 alpha-hydroxy groups of androgens, thereby reactivating androgen metabolites. Finally, it is involved in beta-oxidation of fatty acids by catalyzing the L-hydroxyacyl CoA dehydrogenase reaction of the beta-oxidation cycle. These features and expression profiles suggest a critical role of 17 beta-HSD-10 in neurodegenerative and steroid-dependent cancer forms. Since no three-dimensional structure of 17 beta-HSD-10 is available, homology modelling was carried out to understand the molecular basis of these substrate specificities. The structure obtained displays the properties of a one-domain, alpha/beta fold enzyme of the SDR family. The active site is located within a large, hydrophobic cleft, which forms optimal contacts with the different steroid surfaces. The data provide explanations for the substrate specificities toward the various classes of sex steroid hormones. The model is suitable to explore substrate and inhibitor characteristics that may be used in the development of novel strategies in the treatment of degenerative or malignant diseases.

Bioinformatics in studies of SDR and MDR enzymes.

Bioinformatics utilises information in databases to understand biological processes and interpret experimental data. Methods include sequence comparisons, structural and functional predictions, and molecular modelling. Applied to the families of short-chain and medium-chain dehydrogenases/reductases (SDR and MDR), much information is obtained.

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).

Ancestral diterpene cyclases show increased thermostability and substrate acceptance

Bacterial diterpene cyclases are receiving increasing attention in biocatalysis and synthetic biology for the sustainable generation of complex multicyclic building blocks. Herein, we explore the potential of ancestral sequence reconstruction (ASR) to generate remodeled cyclases with enhanced stability, activity, and promiscuity. Putative ancestors of spiroviolene synthase, a bacterial class I diterpene cyclase, display an increased yield of soluble protein of up to fourfold upon expression in the model organism Escherichia coli. Two of the resurrected enzymes, with an estimated age of approximately 1.7 million years, display an upward shift in thermostability of 7–13 °C. Ancestral spiroviolene synthases catalyze cyclization of the natural C20‐substrate geranylgeranyl diphosphate (GGPP) and also accept C15 farnesyl diphosphate (FPP), which is not converted by the extant enzyme. In contrast, the consensus sequence generated from the corresponding multiple sequence alignment was found to be inactive toward both substrates. Mutation of a nonconserved position within the aspartate‐rich motif of the reconstructed ancestral cyclases was associated with modest effects on activity and relative substrate specificity (i.e., kcat/KM for GGPP over kcat/KM for FPP). Kinetic analyses performed at different temperatures reveal a loss of substrate saturation, when going from the ancestor with highest thermostability to the modern enzyme. The kinetics data also illustrate how an increase in temperature optimum of biocatalysis is reflected in altered entropy and enthalpy of activation. Our findings further highlight the potential and limitations of applying ASR to biosynthetic machineries in secondary metabolism.