Comparative functional analysis of human medium-chain dehydrogenases, short-chain dehydrogenases/reductases and aldo-keto reductases with retinoids

Retinoic acid biosynthesis in vertebrates occurs in two consecutive steps: the oxidation of retinol to retinaldehyde followed by the oxidation of retinaldehyde to retinoic acid. Enzymes of the MDR (medium-chain dehydrogenase/reductase), SDR (short-chain dehydrogenase/reductase) and AKR (aldo-keto reductase) superfamilies have been reported to catalyse the conversion between retinol and retinaldehyde. Estimation of the relative contribution of enzymes of each type was difficult since kinetics were performed with different methodologies, but SDRs would supposedly play a major role because of their low K(m) values, and because they were found to be active with retinol bound to CRBPI (cellular retinol binding protein type I). In the present study we employed detergent-free assays and HPLC-based methodology to characterize side-by-side the retinoid-converting activities of human MDR [ADH (alcohol dehydrogenase) 1B2 and ADH4), SDR (RoDH (retinol dehydrogenase)-4 and RDH11] and AKR (AKR1B1 and AKR1B10) enzymes. Our results demonstrate that none of the enzymes, including the SDR members, are active with CRBPI-bound retinoids, which questions the previously suggested role of CRBPI as a retinol supplier in the retinoic acid synthesis pathway. The members of all three superfamilies exhibit similar and low K(m) values for retinoids (0.12-1.1 microM), whilst they strongly differ in their kcat values, which range from 0.35 min(-1) for AKR1B1 to 302 min(-1) for ADH4. ADHs appear to be more effective retinol dehydrogenases than SDRs because of their higher kcat values, whereas RDH11 and AKR1B10 are efficient retinaldehyde reductases. Cell culture studies support a role for RoDH-4 as a retinol dehydrogenase and for AKR1B1 as a retinaldehyde reductase in vivo.     

Isolation of estrophilic fractions of hydroxysteroid dehydrogenase from rabbit liver

Estrophilic forms of rabbit liver cytosolic hydroxysteroid-dehydrogenase (HSD) were obtained as a highly purified preparations by means of fractionation with ammonium sulfate, gel-filtration, ion-exchange chromatography on DEAE-Sephadex A-50, affinity chromatography on estradiol-Sepharose and ion-exchange chromatography on DEAE-Toyopearl 650M. The protein express 4 different kinds of NADP-dependent activities: 3 alpha, 3 beta- and 17 beta-HSD activities with androgens and 20 alpha-HSD with progesterone as substrates. Revealed multiplicity of HSD enzymatic activity is demonstrated here for the first time. 17 beta-HSD activity of the protein preparations with estradiol is extremely low. Absence of a real metabolic activity of the protein with a ligand interacting with it rather intensively suggests that the isolated HSD forms can act not only as an enzyme, but also as a buffer-reserving mechanism for some steroids.     

Histochemical distribution of hydroxysteroid dehydrogenases in kidney and liver

Summary Mouse, rat, hamster, guinea pig and sheep kidneys and foetal human, adult male and female human, mouse, rat, hamster and guinea pig livers were examined for hydroxysteroid dehydrogenase activity.3-Hydroxysteroids were utilised by all tissues, including neonatal mouse kidney, but the 5-configuration was a more suitable substrate than the corresponding 5-steroid. Both N.A.D. and N.A.D.P. were suitable cofactors.Only trace 3-hydroxysteroid dehydrogenase activity was demonstrable in renal tissue, however liver possessed a higher level of activity and lanosterol, a precurser of cholesterol, was an especially suitable substrate possibly indicating that the liver is capable of synthesising cholesterol.6-Hydroxyprogesterone was poorly utilized by renal and hepatic tissue and N.A.D. was found to be the only cofactor suitable for this reaction. All the tissues, possessed 11-hydroxysteroid dehydrogenase activity. In the kidney, this enzyme occurred in the collecting tubules. It was further noted that in mouse kidney 11-hydroxysteroid dehydrogenase was absent at birth but appeared within the first fourteen days. Activity with 11-hydroxysteroids was observed to be more prominent in the liver of male animals and this pattern was also found with 3-, 3-, 16- and 16-hydroxysteroids, all of which are confirmed by previous biochemical findings.Renal tissue was not capable of utilizing the 16-hydroxysteroid in contrast to liver which could use this substrate fairly well. 16- and 17-hydroxysteroid dehydrogenases were demonstrable in the livers of all species and in all kidneys. The 20-hydroxysteroid was only poorly utilized by hepatic tissue and not at all by renal tissue.Slight activity was demonstrable with 5- and 5-androstans as substrates in liver and the diformazan deposition was presumably due to the action of a steroid reductase.     

pH-dependence of hormone-binding and enzymatic activity of estrophilic hydroxysteroid dehydrogenase from the rabbit liver

The effects of pH on the ability of NADP-dependent estrophilic hydroxysteroid dehydrogenase (HSD) from the soluble fraction of rabbit liver to bind steroids and catalyze their 3 alpha, 3 beta, 17 beta- and 20 alpha-oxidoreduction were studied. The pH optima for enzymatic transformations of various steroids were found to differ significantly by more than two units. These differences do not seem to be related to the localization of the modified group in the steroid molecule. Kinetic data suggest that pH influences the catalytic efficiency, steroid affinity for the protein and, perhaps, the degree of interdependence of steroid and cofactor binding to the protein. These assumptions were confirmed by the results of direct 3H-steroid-HSD binding studies. Furthermore, the maximal levels of binding of various steroids to the protein were found to occur at pH values differing by more than 4 units. Scatchard analysis revealed the effects of hydrogen ion concentrations both on the steroids affinity for the protein and on the concentration of steroid-binding sites of HSD. The data obtained are suggestive of some "superfluity" of the protein steroid-binding site which, in turn, ensures the multifunctionality of estrophilic HSD including a possibility of an alternative orientation of steroids in their binding site.     

Molecular determinants of steroid recognition and catalysis in aldo-keto reductases. Lessons from 3alpha-hydroxysteroid dehydrogenase.

Hydroxysteroid Dehydrogenases (HSDs) regulate the occupancy of steroid hormone receptors by converting active steroid hormones into their cognate inactive metabolites. HSDs belong to either the Short-chain Dehydrogenase/Reductases (SDRs) or the Aldo-Keto Reductases (AKRs). The AKRs include virtually all mammalian 3alpha-HSDs, Type 5 17beta-HSD, ovarian 20alpha-HSDs as well as the steroid 5beta-reductases. Selective inhibitors of 3alpha-HSD isoforms could control occupancy of the androgen and GABA(A) receptors, while broader based AKR inhibitors targeting 3alpha-HSD, 20alpha-HSD and prostaglandin F2alpha synthase could maintain pregnancy. We have determined three X-ray crystal structures of rat liver 3alpha-HSD, a representative AKR. These structures are of the apoenzyme (E), the binary-complex (E.NADP-), and the ternary complex (E.NADP+.testosterone). These structures are being used with site-directed mutagenesis to define the molecular determinants of steroid recognition and catalysis as a first step in rational inhibitor design. A conserved catalytic tetrad (Tyr55, Lys84, His117 and Asp50) participates in a 'proton-relay' in which Tyr55 acts as general acid/base catalyst. Its bifunctionality relies on contributions from His117 and Lys84 which alter the pKb and pKa, respectively of this residue. Point mutation of the tetrad results in different enzymatic activities. H117E mutants display 5beta-reductase activity while Y55F and Y55S mutants retain quinone reductase activity. Our results suggest that different transition states are involved in these reaction mechanisms. The ternary complex structure shows that the mature steroid binding pocket is comprised of ten residues recruited from five loops, and that there is significant movement of a C-terminal loop on binding ligand. Mutagenesis of pocket tryptophans shows that steroid substrates and classes of nonsteroidal inhibitors exhibit different binding modes which may reflect ligand-induced loop movement. Exploitation of these findings using steroidal and nonsteroidal mechanism based inactivators may lead to selective and broad based AKR inhibitors.     

Mechanism based inhibition of hydroxysteroid dehydrogenases.

Steroid hormone action can be regulated not only at the receptor level but also by the enzymes that are responsible for the synthesis and degradation of biologically active steroids. Traditionally the pharmacological intervention of steroid hormone action has focused on the development of steroidal and nonsteroidal hormone receptor agonists and antagonists with appropriate pharmacokinetics. Recently, the development of selective inhibitors/inactivators of steroid metabolizing enzymes has gained momentum. This review will concentrate on the development of mechanism-based inhibitors for one class of steroid hormone transforming enzymes, the hydroxysteroid dehydrogenases.     

The effect of cofactors on the binding of steroid substrates with extrophilic hydroxysteroid dehydrogenase in the rabbit liver

The isolated NADP (H)-dependent extrophilic hydroxysteroid dehydrogenase (EHSD) catalyzes oxidative-reductive reactions of 3 alpha-, 3 beta-, 17 beta-, and 20 alpha-hydroxy groups of androgens and progestagens, and binds these steroids and estrogens with relatively high affinities. In three series of experiments of the enzymatic kinetics, binding of 3H steroids to the protein, and inhibitory analysis an influence of cofactors on the affinity of steroid substrates and their analogs to the protein was demonstrated. A degree and direction of this influence are dependent on the steroid structure and cofactor form. It is proposed that cofactors may be important physiological regulators of enzymatic and steromoduline functions of the isolated EHSD.     

Substrate specificity and catalytic efficiency of aldo-keto reductases with phospholipid aldehydes

Phospholipid oxidation generates several bioactive aldehydes that remain esterified to the glycerol backbone ('core' aldehydes). These aldehydes induce endothelial cells to produce monocyte chemotactic factors and enhance monocyte-endothelium adhesion. They also serve as ligands of scavenger receptors for the uptake of oxidized lipoproteins or apoptotic cells. The biochemical pathways involved in phospholipid aldehyde metabolism, however, remain largely unknown. In the present study, we have examined the efficacy of the three mammalian AKR (aldo-keto reductase) families in catalysing the reduction of phospholipid aldehydes. The model phospholipid aldehyde POVPC [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphocholine] was efficiently reduced by members of the AKR1, but not by the AKR6 or the ARK7 family. In the AKR1 family, POVPC reductase activity was limited to AKR1A and B. No significant activity was observed with AKR1C enzymes. Among the active proteins, human AR (aldose reductase) (AKR1B1) showed the highest catalytic activity. The catalytic efficiency of human small intestinal AR (AKR1B10) was comparable with the murine AKR1B proteins 1B3 and 1B8. Among the murine proteins AKR1A4 and AKR1B7 showed appreciably lower catalytic activity as compared with 1B3 and 1B8. The human AKRs, 1B1 and 1B10, and the murine proteins, 1B3 and 1B8, also reduced C-7 and C-9 sn-2 aldehydes as well as POVPE [1-palmitoyl-2-(5-oxovaleroyl)-sn-glycero-3-phosphoethanolamine]. AKR1A4, B1, B7 and B8 catalysed the reduction of aldehydes generated in oxidized C(16:0-20:4) phosphatidylcholine with acyl, plasmenyl or alkyl linkage at the sn-1 position or C(16:0-20:4) phosphatidylglycerol or phosphatidic acid. AKR1B1 displayed the highest activity with phosphatidic acids; AKR1A4 was more efficient with long-chain aldehydes such as 5-hydroxy-8-oxo-6-octenoyl derivatives, whereas AKR1B8 preferred phosphatidylglycerol. These results suggest that proteins of the AKR1A and B families are efficient phospholipid aldehyde reductases, with non-overlapping substrate specificity, and may be involved in tissue-specific metabolism of endogenous or dietary phospholipid aldehydes.     

Continuous-flow automated assay of steroids with nylon-tube-immobilized hydroxysteroid dehydrogenases

Several NAD(P)⁺-dependent hydroxysteroid dehydrogenases, namely 3α-hydroxysteroid dehydrogenase, β-hydroxysteroid dehydrogenase, 7α-hydroxysteroid dehydrogenase, and 12α-hydroxysteroid dehydrogenase were separately immobilized on nylon tubes for the continuous-flow automated assay of hydroxysteroids. 3α-Hydroxysteroid dehydrogenase was also immobilized on pore glass. Spectrophotometric monitoring in the visible region, where blank values were markedly reduced, was achieved through the Meldola blue catalyzed transfer of hydrogen from NAD(P)H to a tetrazolium salt. Nylon-tube-immobilized enzymes maintained 45–55% of the original activity after 1 month of intermittent use. The operational range, using the “end point” approach, was 1–25 nmol of steroid and the assay speed 10–15 samples/h. Reliable results were obtained in the determination of 3α-hydroxysteroids and 3β,17β-hydroxysteroids in urine and total bile acids in serum.     

9,10-Phenanthrenequinone promotes secretion of pulmonary aldo-keto reductases with surfactant

9,10-Phenanthrenequinone (9,10-PQ), a major quinone in diesel exhaust particles, induces apoptosis via the generation of reactive oxygen species (ROS) because of 9,10-PQ redox cycling. We have found that intratracheal infusion of 9,10-PQ facilitates the secretion of surfactant into rat alveolus. In the cultured rat lung, treatment with 9,10-PQ results in an increase in a lower-density surfactant by ROS generation through redox cycling of the quinone. The surfactant contains aldo-keto reductase (AKR) 1C15, which reduces 9,10-PQ and the enzyme level in the surfactant increases on treatment with 9,10-PQ suggesting an involvement of AKR1C15 in the redox cycling of the quinone. In six human cell types (A549, MKN45, Caco2, Hela, Molt4 and U937) only type II epithelial A549 cells secrete three human AKR1C subfamily members (AKR1C1, AKR1C2 and AKR1C3) with the surfactant into the medium; this secretion is highly increased by 9,10-PQ treatment. Using in vitro enzyme inhibition analysis, we have identified AKR1C3 as the most abundantly secreted AKR1C member. The AKR1C enzymes in the medium efficiently reduce 9,10-PQ and initiate its redox cycling accompanied by ROS production. The exposure of A549 cells to 9,10-PQ provokes viability loss, which is significantly protected by the addition of the AKR1C3 inhibitor and antioxidant enzyme and by the removal of the surfactants from the culture medium. Thus, the AKR1C enzymes secreted in pulmonary surfactants probably participate in the toxic mechanism triggered by 9,10-PQ.     

Guinea pig liver aromatic aldehyde-ketone reductases identical with 17 beta-hydroxysteroid dehydrogenase isozymes.

Two NADPH-dependent aromatic aldehyde-ketone reductases purified from guinea pig liver catalyzed oxidoreduction of 17 beta-hydroxysteroids and 17-ketosteroids. One enzyme efficiently oxidized 5 beta-androstanes and reduced 17-ketosteroids of A/B cis configuration, whereas the other enzyme efficiently oxidized 5 alpha-androstanes and equally reduced both 5 alpha-and 5 beta-androstanes of 17-ketosteroids. However, aromatic aldehydes and ketones, and 3-ketosteroids were irreversibly reduced by the two enzymes. The two enzymes utilized NADP+ or NADPH as cofactor, but little activity with NAD+ or NADH was found. Phosphate ions enhanced the NAD+-dependent dehydrogenase activity and NADH-dependent reductase activity of the two enzymes, whereas the activities with NADP+ and NADPH were not affected. The ratios of the two activities of ketone reduction and 17 beta-hydroxysteroid oxidation of the two enzymes were almost constant during the purification steps after the two enzymes had been separated by DEAE-cellulose chromatography. By kinetic studies and electrophoresis and isoelectric focusing experiments it was confirmed that both of the two enzymes were responsile for the reduction aldehydes, ketones, and ketosteroids and for the oxidation of 17 beta-hydroxysteroids. These results indicate that 17 beta-hydroxysteroid dehydrogenases may play important roles in the metabolism of exogeneous aldehydes and ketones as well as steroids.     

Dietary and hormonal regulation of some enzyme activities associated with gluconeogenesis in rabbit liver.

1. Starvation increases the activity of cytosolic P-enolpyruvate carboxkinase in rabbit liver some 4-5 fold but does not alter the activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase or glucose-6-phosphatase.2. Alloxan-induced diabetes increases the activities of cytosolic P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase approx. 6-, 2- and 2-fold, respectively. Again the activity of mitochondrial P-enolpyruvate carboxykinase is not altered. 3. Administration of mannoheptulose rapidly increases blood glucose levels and also causes a significant increase in cytosolic P-enolpyruvate carboyxkinase activity within 4 h. The activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase are not affected. 4. Administration of hydrocortisone also increases blood glucose levels and the activities of cytosolic P-enolpyruvate carboxykinase and glucose-6-phosphatase are significantly increased within 12h. Again, mitochondrial P-enolpyruvate carboxykinase and fructose-1,6-diphosphatase activities remain unaffected. 5. The observations that (A) the activity of cytosolic P-enolpyruvate carboxykinase responds to more situations conducive to gluconeogenesis than do the activities of mitochondrial P-enolpyruvate carboxykinase, fructose-1,6-diphosphatase and glucose-6-phosphatase, and (B) cytosolic P-enolpyruvate carboxykinase activity is rapidly adaptive under appropriate circumstances, suggests that this particular enzyme's activity plays an important role in the regulation of gluconeogenesis in rabbits.     

Halohydrin dehalogenases are structurally and mechanistically related to short-chain dehydrogenases/reductases

Halohydrin dehalogenases, also known as haloalcohol dehalogenases or halohydrin hydrogen-halide lyases, catalyze the nucleophilic displacement of a halogen by a vicinal hydroxyl function in halohydrins to yield epoxides. Three novel bacterial genes encoding halohydrin dehalogenases were cloned and expressed in Escherichia coli, and the enzymes were shown to display remarkable differences in substrate specificity. The halohydrin dehalogenase of Agrobacterium radiobacter strain AD1, designated HheC, was purified to homogeneity. The k(cat) and K(m) values of this 28-kDa protein with 1,3-dichloro-2-propanol were 37 s(-1) and 0.010 mM, respectively. A sequence homology search as well as secondary and tertiary structure predictions indicated that the halohydrin dehalogenases are structurally similar to proteins belonging to the family of short-chain dehydrogenases/reductases (SDRs). Moreover, catalytically important serine and tyrosine residues that are highly conserved in the SDR family are also present in HheC and other halohydrin dehalogenases. The third essential catalytic residue in the SDR family, a lysine, is replaced by an arginine in halohydrin dehalogenases. A site-directed mutagenesis study, with HheC as a model enzyme, supports a mechanism for halohydrin dehalogenases in which the conserved Tyr145 acts as a catalytic base and Ser132 is involved in substrate binding. The primary role of Arg149 may be lowering of the pK(a) of Tyr145, which abstracts a proton from the substrate hydroxyl group to increase its nucleophilicity for displacement of the neighboring halide. The proposed mechanism is fundamentally different from that of the well-studied hydrolytic dehalogenases, since it does not involve a covalent enzyme-substrate intermediate.     

Characterization of bovine liver cytosolic 3 alpha-hydroxysteroid dehydrogenase and its aldo-keto reductase activity.

1. 3 alpha-Hydroxysteroid dehydrogenase was purified to homogeneity from bovine cytosolic fraction, which was monomeric and its molecular weight was estimated to be about 35 kDa. 2. The enzyme had ability to catalyze NADP(H)-dependent oxidoreduction of position 3 alpha-hydroxy and keto group of steroids and also could catalyze the reduction of some ketones and quinones. 3. In addition, benzenedihydrodiol was one of the substrates of dehydrogenase activity with NADP+. 4. Indomethacin, synthetic steroids and SH-reagents were potent inhibitors for this enzyme. 5. Inactivation of the enzyme by GSSG-treatment was restored to its original activity by the addition of DTT. 6. The presence of coenzyme, 0.33 mM NADP+, completely protected from the DTNB-inactivation. 7. Bovine liver cytosolic enzyme immunologically crossreacted with rat liver 3 alpha-hydroxysteroid dehydrogenase.     

A cluster of eight hydroxysteroid dehydrogenase genes belonging to the aldo-keto reductase supergene family on mouse chromosome 13

A subclass of hydroxysteroid dehydrogenases (HSD) are NADP(H)-dependent oxidoreductases that belong to the aldo-keto reductase (AKR) superfamily. They are involved in prereceptor or intracrine steroid modulation, and also act as bile acid-binding proteins. The HSD family members characterized thus far in human and rat have a high degree of protein sequence similarity but exhibit distinct substrate specificity. Here we report the identification of nine murine AKR genes in a cluster on chromosome 13 by a combination of molecular cloning and in silico analysis of this region. These include four previously isolated mouse HSD genes (Akr1c18, Akr1c6, Akr1c12, Akr1c13), the more distantly related Akr1e1, and four novel HSD genes. These genes exhibit highly conserved exon/intron organization and protein sequence predictions indicate 75% amino acid similarity. The previously identified AKR protein active site residues are invariant among all nine proteins, but differences are observed in regions that have been implicated in determining substrate specificity. Differences also occur in tissue expression patterns, with expression of some genes restricted to specific tissues and others expressed at high levels in multiple tissues. Our findings dramatically expand the repertoire of AKR genes and identify unrecognized family members with potential roles in the regulation of steroid metabolism.     

Isolation of multiple forms of indanol dehydrogenase associated with 17 beta-hydroxysteroid dehydrogenase activity from male rabbit liver

Seven multiforms of indanol dehydrogenase were isolated in a highly purified state from male rabbit liver cytosol. The enzymes were monomeric proteins with similar molecular weights of 30,000-37,000 but with distinct electrophoretic mobilities. All the enzymes oxidized alicyclic alcohols including benzene dihydrodiol and hydroxysteroids at different optimal pH, but showed clear differences in cofactor specificity, steroid specificity, and reversibility of the reaction. Two NADP+-dependent enzymes exhibited both 17 beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes and 3 alpha-hydroxysteroid dehydrogenase activity for 5 beta-androstan-3 alpha-ol-17-one. Three of the other enzymes with dual cofactor specificity catalyzed predominantly 5 beta-androstane-3 alpha,17 beta-diol dehydrogenation. The reverse reaction rates of these five enzymes were low, whereas the other two enzymes, which had 3 alpha-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes or 3(17)beta-hydroxysteroid dehydrogenase activity for 5 alpha-androstanes, highly reduced 3-ketosteroids and nonsteroidal aromatic carbonyl compounds with NADPH as a cofactor. All the enzymes exhibited Km values lower for the hydroxysteroids than for the alicyclic alcohols. The results of kinetic analyses with a mixture of 1-indanol and hydroxysteroids, pH and heat stability, and inhibitor sensitivity suggested strongly that, in the seven enzymes, both alicyclic alcohol dehydrogenase and hydroxysteroid dehydrogenase activities reside on a single enzyme protein. On the basis of these data, we suggest that indanol dehydrogenase exists in multiple forms in rabbit liver cytosol and may function in in vivo androgen metabolism.