Overview of human 20 alpha-hydroxysteroid dehydrogenase (AKR1C1): Functions, regulation, and structural insights of inhibitors.

Human aldo-keto reductase family 1C1 (AKR1C1) is an important enzyme involved in human hormone metabolism, which is mainly responsible for the metabolism of progesterone in the human body. AKR1C1 is highly expressed and has an important relationship with the occurrence and development of various diseases, especially some cancers related to hormone metabolism. Nowadays, many inhibitors against AKR1C1 have been discovered, including some synthetic compounds and natural products, which have certain inhibitory activity against AKR1C1 at the target level. Here we briefly reviewed the physiological and pathological functions of AKR1C1 and the relationship with the disease, and then summarized the development of AKR1C1 inhibitors, elucidated the interaction between inhibitors and AKR1C1 through molecular docking results and existing co-crystal structures. Finally, we discussed the design ideals of selective AKR1C1 inhibitors from the perspective of AKR1C1 structure, discussed the prospects of AKR1C1 in the treatment of human diseases in terms of biomarkers, pre-receptor regulation and single nucleotide polymorphisms, aiming to provide new ideas for drug research targeting AKR1C1.

Aldo-Keto Reductase 1C1 (AKR1C1) as the First Mutated Gene in a Family with Nonsyndromic Primary Lipedema.

Lipedema is an often underdiagnosed chronic disorder that affects subcutaneous adipose tissue almost exclusively in women, which leads to disproportionate fat accumulation in the lower and upper body extremities. Common comorbidities include anxiety, depression, and pain. The correlation between mood disorder and subcutaneous fat deposition suggests the involvement of steroids metabolism and neurohormones signaling, however no clear association has been established so far. In this study, we report on a family with three patients affected by sex-limited autosomal dominant nonsyndromic lipedema. They had been screened by whole exome sequencing (WES) which led to the discovery of a missense variant p.(Leu213Gln) in AKR1C1, the gene encoding for an aldo-keto reductase catalyzing the reduction of progesterone to its inactive form, 20-α-hydroxyprogesterone. Comparative molecular dynamics simulations of the wild-type vs. variant enzyme, corroborated by a thorough structural and functional bioinformatic analysis, suggest a partial loss-of-function of the variant. This would result in a slower and less efficient reduction of progesterone to hydroxyprogesterone and an increased subcutaneous fat deposition in variant carriers. Overall, our results suggest that AKR1C1 is the first candidate gene associated with nonsyndromic lipedema.

The SIRT2-mediated deacetylation of AKR1C1 is required for suppressing its pro-metastasis function in Non-Small Cell Lung Cancer.

Aldo-keto reductase family 1 member C1 (AKR1C1) promotes malignancy of Non-Small Cell Lung Cancer (NSCLC) by activating Signal Transducer and Activator of Transcription 3 (STAT3) pathway. However, how the pro-metastatic functions of AKR1C1 are switched on/off remains unknown. Methods: Immunoprecipitation and LC-MS/MS analyses were performed to identify the acetylation on AKR1C1 protein, and the functional analyses (in vitro and in vivo) were performed to depict the contribution of acetylation to the pro-metastatic effects of AKR1C1. Results: Here we report that acetylated AKR1C1 on two lysine residues K185 & K201 is critical to its pro-metastatic role. The acetylation modification has no impact on the canonical enzymatic activity of AKR1C1, while it is required for the interaction between AKR1C1 to STAT3, which triggers the downstream transduction events, ultimately mobilizing cells. Importantly, the deacetylase Sirtuin 2 (SIRT2) is capable of deacetylating AKR1C1, inhibiting the transactivation of STAT3 target genes, thus suppressing the migration of cells. Conclusion: Acetylation on Lysines 185 and 201 of AKR1C1 dictates its pro-metastatic potential both in vitro and in vivo, and the reverting of acetylation by Sirtuin 2 provides potential therapeutic targets for treatment against metastatic NSCLC patients with high AKR1C1 expression.

Human dehydrogenase/reductase SDR family member 11 (DHRS11) and aldo-keto reductase 1C isoforms in comparison: Substrate and reaction specificity in the reduction of 11-keto-C19-steroids.

Recent studies have shown that an adrenal steroid 11ß-hydroxy-4-androstene-3,17-dione serves as the precursor to androgens, 11-ketotestosterone and 11-ketodihydrotestosterone (11KDHT). The biosynthetic pathways include the reduction of 3- and 17-keto groups of the androgen precursors 11-keto-C19-steroids, which has been reported to be mediated by three human enzymes; aldo-keto reductase (AKR)1C2, AKR1C3 and 17ß-hydroxysteroid dehydrogenase (HSD) type-3. To explore the contribution of the enzymes in the reductive metabolism, we kinetically compared the substrate specificity for 11-keto-C19-steroids among purified recombinant preparations of four AKRs (1C1, 1C2,1C3 and 1C4) and DHRS11, which shows 17ß-HSD activity. Although AKR1C1 did not reduce the 11-keto-C19-steroids, AKR1C3 and DHRS11 reduced 17-keto groups of 11-keto-4-androstene-3,17-dione, 11-keto-5α-androstane-3,17-dione (11K-Adione) and 11-ketoandrosterone with Km values of 5-28 µM. The 3-keto groups of 11KDHT and 11K-Adione were reduced by AKR1C4 (Km 1 µM) more efficiently than by AKR1C2 (Km 5 and 8 µM, respectively). GC/MS analysis of the products showed that DHRS11 acts as 17ß-HSD, and that AKR1C2 and AKR1C4 are predominantly 3α-HSDs, but formed a minor 3ß-metabolite from 11KDHT. Since DHRS11 was thus newly identified as 11-keto-C19-steroid reductase, we also investigated its substrate-binding mode by molecular docking and site-directed mutagenesis of Thr163 and Val200, and found the following structural features: 1). There is a space that accommodates the 11-keto group of the 11-keto-C19-steroids in the substrate-binding site. 2) Val200 is a critical determinant for exhibiting the strict 17ß-HSD activity of the enzyme, because the Val200Leu mutation resulted in both significant impairment of the 17ß-HSD activity and emergence of 3ß-HSD activity towards 5α-androstanes including 11KDHT.

Molecular Docking, Synthesis and Biological Evaluation of Sulphonylureas/ Guanidine Derivatives as Promising Antidiabetic Agent.

BACKGROUND: A series of novel sulphonylureas/guanidine derivatives was designed, synthesized, and evaluated for the treatment of diabetes mellitus. In this study, the designed compounds were docked with AKR1C1 complexes by using glide docking program and docking calculations were performed to predict the binding affinity of the designed compounds with the binding pocket of protein 4YVP and QikProp program was used to predict the ADME/T properties of the analogues. METHODS: All the targeted derivatives were synthesized and purified by recrystallization. Synthesized compounds were characterized by various physicochemical and various spectroscopic techniques like melting point, thin layer chromatography, infrared spectroscopy (KBr pellets), mass spectroscopy(m/z), 1H NMR (DMSO-d6), and 13C NMR. The synthesized compounds were further studied for biological evolution by alloxan (150 mg/dl, intraperitonial) induced diabetic rat model for in-vivo studies. RESULT: Among all the synthesized derivatives, 5c and 5d were most potent as per binding energy. Compound 5i have shown a better plasma glucose reduction compared to glibenclamide. Hence, it will be further used as a lead compound to develop a more such kind of agent. CONCLUSION: The docking study revealed that in all designed sulphonylureas/ guanidine series of compounds 5c and 5d were found to be most potent compounds as per the binding energy compared to glibenclamide. With the help of detailed study of in vivo biological activity, we observed that compound 5i gives better result compared to glibenclamide as standard.

Structural importance of the C-terminal region in pig aldo-keto reductase family 1 member C1 and their effects on enzymatic activity.

BACKGROUND: Pig aldo-keto reductase family 1 member C1 (AKR1C1) belongs to AKR superfamily which catalyzes the NAD(P)H-dependent reduction of various substrates including steroid hormones. Previously we have reported two paralogous pig AKR1C1s, wild-type AKR1C1 (C-type) and C-terminal-truncated AKR1C1 (T-type). Also, the C-terminal region significantly contributes to the NADPH-dependent reductase activity for 5α-DHT reduction. Molecular modeling studies combined with kinetic experiments were performed to investigate structural and enzymatic differences between wild-type AKR1C1 C-type and T-type. RESULTS: The results of the enzyme kinetics revealed that Vmax and kcat values of the T-type were 2.9 and 1.6 folds higher than those of the C-type. Moreover, catalytic efficiency was also 1.9 fold higher in T-type compared to C-type. Since x-ray crystal structures of pig AKR1C1 were not available, three dimensional structures of the both types of the protein were predicted using homology modeling methodology and they were used for molecular dynamics simulations. The structural comparisons between C-type and T-type showed that 5α-DHT formed strong hydrogen bonds with catalytic residues such as Tyr55 and His117 in T-type. In particular, C3 ketone group of the substrate was close to Tyr55 and NADPH in T-type. CONCLUSIONS: Our results showed that 5α-DHT binding in T-type was more favorable for catalytic reaction to facilitate hydride transfer from the cofactor, and were consistent with experimental results. We believe that our study provides valuable information to understand important role of C-terminal region that affects enzymatic properties for 5α-DHT, and further molecular mechanism for the enzyme kinetics of AKR1C1 proteins.

Important role of the C-terminal region of pig aldo-keto reductase family 1 member C1 in the NADPH-dependent reduction of steroid hormones.

The NADPH-dependent reduction activities of two paralogous pig AKR1C1s with and without 19 additional amino acid residues in C-terminus were evaluated against steroid hormones including 5alpha-dihydrotestosterone, testosterone, progesterone, androstenedione and 5alpha-androstane-3.17-dione, which act as substrates of the AKR1C1S. Among the hormones, the AKR1C1s exhibited the highest activity against 5alpha-dihydrotestosterone and the lowest activity against testosterone and progesterone. Furthermore, the AKR1C1s showed the largest differential activities against; 5alpha-dihydrotestosterone, but no such change of activities was found against progestrone and testosterone. These results suggest that the C-terminal region of AKR1C1 plays an important effect in the reduction activities of pig AKR1C1. Thus, the differential activities of two AKR1C1 paralogs observed in the present study provide important insights in understanding the molecular evolution.

Selective inhibitors of aldo-keto reductases AKR1C1 and AKR1C3 discovered by virtual screening of a fragment library.

Human aldo-keto reductases 1C1-1C4 (AKR1C1-AKR1C4) function in vivo as 3-keto-, 17-keto-, and 20-ketosteroid reductases and regulate the activity of androgens, estrogens, and progesterone and the occupancy and transactivation of their corresponding receptors. Aberrant expression and action of AKR1C enzymes can lead to different pathophysiological conditions. AKR1C enzymes thus represent important targets for development of new drugs. We performed a virtual high-throughput screen of a fragment library that was followed by biochemical evaluation on AKR1C1-AKR1C4 enzymes. Twenty-four structurally diverse compounds were discovered with low µM K(i) values for AKR1C1, AKR1C3, or both. Two structural series included the salicylates and the N-phenylanthranilic acids, and additionally a series of inhibitors with completely novel scaffolds was discovered. Two of the best selective AKR1C3 inhibitors had K(i) values of 0.1 and 2.7 µM, exceeding expected activity for fragments. The compounds identified represent an excellent starting point for further hit-to-lead development.

Ovarian and placental expression of 20α-hydroxysteroid dehydrogenase during pregnancy in deer.

The enzyme 20α-hydroxysteroid dehydrogenase (20α-HSD) catalyzes the conversion of progesterone to its inactive form, 20α-hydroxyprogesterone. This enzyme has been shown to play a critical role in the regulation of luteal function in experimental animals. In this study, we cloned and expressed the gene encoding elk deer 20α-HSD from reproductive placental and ovarian tissues. PCR, 3'- and 5'-RACE, and northern blot analysis were performed for the cloning and characterization of deer 20α-HSD gene. We expressed recombinant deer 20α-HSD protein and used western blot analysis to determine protein expression levels in the placenta and ovary during pregnancy. The full cDNA sequence of 20α-HSD was used to clone an open reading frame encoding 323 amino acids and consisting of 1142 bp. The nucleotide sequence of deer 20α-HSD showed high homology with the sequences of the bovine (96%), goat (96%), and human (83%) 20α-HSD genes. 20α-HSD mRNA was strongly expressed in the placenta on days 30, 60, and 70 of pregnancy. A high level of the protein was also detected in the placenta but not in fetal skin tissue. The recombinant 20α-HSD protein produced in mammalian cells and bacterial systems had a molecular weight of approximately 37-kDa. The deer 20α-HSD protein signal was specifically localized in the basal part of the primary chorionic villi and chorionic stem villus of the placenta during early pregnancy. The 20α-HSD protein was also intensively localized in the larger luteal cells of the corpus luteum during pregnancy.

Retinaldehyde is a substrate for human aldo-keto reductases of the 1C subfamily.

Human AKR (aldo-keto reductase) 1C proteins (AKR1C1-AKR1C4) exhibit relevant activity with steroids, regulating hormone signalling at the pre-receptor level. In the present study, investigate the activity of the four human AKR1C enzymes with retinol and retinaldehyde. All of the enzymes except AKR1C2 showed retinaldehyde reductase activity with low Km values (~1 µM). The kcat values were also low (0.18-0.6 min-1), except for AKR1C3 reduction of 9-cis-retinaldehyde whose kcat was remarkably higher (13 min-1). Structural modelling of the AKR1C complexes with 9-cis-retinaldehyde indicated a distinct conformation of Trp227, caused by changes in residue 226 that may contribute to the activity differences observed. This was partially supported by the kinetics of the AKR1C3 R226P mutant. Retinol/retinaldehyde conversion, combined with the use of the inhibitor flufenamic acid, indicated a relevant role for endogenous AKR1Cs in retinaldehyde reduction in MCF-7 breast cancer cells. Overexpression of AKR1C proteins depleted RA (retinoic acid) transactivation in HeLa cells treated with retinol. Thus AKR1Cs may decrease RA levels in vivo. Finally, by using lithocholic acid as an AKR1C3 inhibitor and UVI2024 as an RA receptor antagonist, we provide evidence that the pro-proliferative action of AKR1C3 in HL-60 cells involves the RA signalling pathway and that this is in part due to the retinaldehyde reductase activity of AKR1C3.

Stereospecific reduction of 5ß-reduced steroids by human ketosteroid reductases of the AKR (aldo-keto reductase) superfamily: role of AKR1C1-AKR1C4 in the metabolism of testosterone and progesterone via the 5ß-reductase pathway.

Active sex hormones such as testosterone and progesterone are metabolized to tetrahydrosteroids in the liver to terminate hormone action. One main metabolic pathway, the 5ß-pathway, involves 5ß-steroid reductase (AKR1D1, where AKR refers to the aldo-keto reductase superfamily), which catalyses the reduction of the 4-ene structure, and ketosteroid reductases (AKR1C1-AKR1C4), which catalyse the subsequent reduction of the 3-oxo group. The activities of the four human AKR1C enzymes on 5ß-dihydrotestosterone, 5ß-pregnane-3,20-dione and 20α-hydroxy-5ß-pregnan-3-one, the intermediate 5ß-dihydrosteroids on the 5ß-pathway of testosterone and progesterone metabolism, were investigated. Product characterization by liquid chromatography-MS revealed that the reduction of the 3-oxo group of the three steroids predominantly favoured the formation of the corresponding 3α-hydroxy steroids. The stereochemistry was explained by molecular docking. Kinetic properties of the enzymes identified AKR1C4 as the major enzyme responsible for the hepatic formation of 5ß-tetrahydrosteroid of testosterone, but indicated differential routes and roles of human AKR1C for the hepatic formation of 5ß-tetrahydrosteroids of progesterone. Comparison of the kinetics of the AKR1C1-AKR1C4-catalysed reactions with those of AKR1D1 suggested that the three intermediate 5ß-dihydrosteroids derived from testosterone and progesterone are unlikely to accumulate in liver, and that the identities and levels of 5ß-reduced metabolites formed in peripheral tissues will be governed by the local expression of AKR1D1 and AKR1C1-AKR1C3.

Cloning, characterization, sequence analysis and expression patterns in vivo of testicular 20ß-hydroxysteroid dehydrogenase cDNA in yellow catfish (Pelteobagrus fulvidraco).

We have cloned a full-length cDNA for testicular 20ß-HSD in yellow catfish. The validated 20ß-HSD cDNA full-length sequence, 1141 bp in length, contained a 108 bp 5'-untranslated region (UTR), a 202 bp 3'-UTR with an AATAAAA frame, and an 831 bp open reading frame (ORF) which encoded a propeptide of 277 amino acid residues. The enzyme shows the highest structural homology with that of zebrafish, and rainbow trout. Quantitative real-time PCR revealed that 20ß-HSD has widespread tissue distribution, with expression being abundant in tissues with high metabolic rates like gonads, liver, intestine, stomach and gill. In vivo experiments showed that expression level was highest at testicular mature stage indicating that 20ß-HSD could play an important role in testicular developmental maturation in yellow catfish. During testicular mature stage, 20ß-HSD related metabolism was regulated by GnRH and LH. Moreover, structural analysis showed that the predicted 20ß-HSD contained 7 functional motifs of SDR superfamily of enzymes, including the putative coenzyme binding domain (Rossmann fold), GlyXXXGlyIleuGly, and the region responsible for nucleophilic attack of the substrate pocket, TyrXXXLys. These motifs are strictly conserved in yellow catfish 20ß-HSD. Comprehensive functional analysis revealed that this enzyme has multiple functions, such as xenobiotic metabolism, and steroid conversion. Catfish 20ß-HSD contains multiple potential post-translational modification sites. Its subcellular location, theoretical isoelectric point and molecular weight were also investigated. Furthermore, we constructed its phylogenetic tree and secondary structure. All results provided basic information for further studies of its structure, functions and properties.

Inhibitors of human 20α-hydroxysteroid dehydrogenase (AKR1C1).

Human 20α-hydroxysteroid dehydrogenase (AKR1C1), a member of the aldo-keto reductase (AKR) superfamily, is one of four isoforms (with >84% amino acid sequence identity) existing in human tissues. AKR1C1 most efficiently reduces biologically active progesterone and 5α-pregnan-3α-ol-20-one into their corresponding 20α-hydroxysteroids among the isoforms. The enzyme also accepts endogenous and xenobiotic non-steroidal carbonyl compounds as the substrates. In addition to the up-regulation of the AKR1C1 gene in cancer cells, the enzyme's over-expression in the cells of lung, ovary, uterine cervix, skin and colon carcinomas was reported to be associated with resistance against several anticancer agents. Thus, AKR1C1 may be a marker of the above cancers and a target of poor prognosis in cancer therapy. The recently determined X-ray crystal structures of AKR1C1/NADP(+)/20α-hydroxyprogesterone and AKR1C1/NADP(+)/3,5-dichlorosalicylic acid ternary complexes have provided a strong foundation for structure-based design methods to improve inhibitor selectivity and potency. In this review we provide an overview of the different types of AKR1C1 inhibitors and an update on the design of potent and selective inhibitors based on the crystal structure of the enzyme-inhibitor complex. Article from the Special issue on Targeted Inhibitors.

Progestins as inhibitors of the human 20-ketosteroid reductases, AKR1C1 and AKR1C3.

The human aldo-keto reductases 1C1 and 1C3 (AKR1C1 and AKR1C3) are important 20-ketosteroid reductases in pre-receptor regulation of progesterone action. Both AKR1C1 and AKR1C3 convert progesterone to the less potent metabolite 20α-hydroxyprogesterone, although AKR1C1 has a higher catalytic efficiency than AKR1C3. Recently, we reported significant up-regulation of AKR1C1 and AKR1C3 in ovarian endometriosis, a complex estrogen-dependent disease. The typical characteristics of endometriosis are increased formation of estradiol, which stimulates proliferation of endometriotic tissue, and disturbed action of the protective progesterone. Although progestins have been used for treatment of endometriosis since the 1960s, their detailed mechanisms of action are still not completely understood. In the present study, we evaluated the potential inhibitory effects of progestins on the pre-receptor regulatory enzymes AKR1C1 and AKR1C3. We examined the following progestins as inhibitors of progesterone reduction catalyzed by recombinant AKR1C1 and AKR1C3: progesterone derivatives (dydrogesterone, its metabolite, 20α-hydroxydydrogesterone; and medroxyprogesterone acetate), 19-nortestosterone derivatives (desogestrel, norethinodrone and levonorgestrel), and the androgen danazol. Dydrogesterone, medroxyprogesterone acetate, 20α-hydroxydydrogesterone and norethinodrone inhibited AKR1C1 and AKR1C3 with K(i) values of 1.9 µM, 7.9 µM, 20.8 µM and 48.0 µM, and of 0.5 µM, 1.4 µM, 18.2 µM and 6.6 µM, respectively. Levonorgestrel and desogestrel preferentially inhibited AKR1C3 with K(i) values of 5.6µM and 39.1µM, respectively. Our data thus show that dydrogesterone, medroxyprogesterone acetate, 20α-hydroxydydrogesterone and norethinodrone inhibit AKR1C1 and AKR1C3 in vitro, although their physiological inhibitory effects still need to be evaluated further. Additionally, we investigated whether progestin dydrogesterone can be metabolized to its active 20α-hydroxymetabolite by AKR1C1 and AKR1C3. AKR1C1 converted dydrogesterone with a high catalytic efficiency while AKR1C3 was less active, which suggests that in vivo dydrogesterone is metabolized mainly by AKR1C1. Docking simulations of dydrogesterone into AKR1C1 and AKR1C3 also support these experimental data.

Human 20α-hydroxysteroid dehydrogenase (AKR1C1)-dependent biotransformation with recombinant fission yeast Schizosaccharomyces pombe.

While phase I and phase II drug metabolites are important for drug development and toxicity studies, e.g. in the context of metabolites in safety testing (MIST), they are often not commercially available and their classical chemical synthesis can be cumbersome. Therefore, a biotechnological production of drug metabolites using microorganisms that recombinantly express human enzymes has been established in recent years. However, no whole-cell biotransformations that make use of human aldo-keto reductases (AKRs) have yet been reported. In this study, we have functionally expressed human AKR1C1 (20α-hydroxysteroid dehydrogenase) in the fission yeast Schizosaccharomyces pombe and demonstrate the ability of the resulting yeast strain to efficiently catalyze the reduction of progesterone or dydrogesterone to 20α-dihydroprogesterone (20α-DHP) and 20α-dihydrodydrogesterone (20α-DHD), respectively. The formation of any by-products or the occurrence of a back reaction were not detected. Seven other steroids with a 20-keto group (pregnenolone, 17α-hydroxyprogesterone, 11-deoxycortisol, cortisol, 11-deoxycorticosterone, corticosterone, and aldosterone) were not reduced by this system. At shaking flask scale we obtained conversion rates of 90 (±26) µM/d 20α-DHP and 244 (±93) µM/d 20α-dihydrodydrogesterone (20α-DHD), respectively. In a fed-batch fermentation under optimized reaction conditions an average 20α-DHP production rate of 300 µM/d was determined for a total biotransformation time of 72 h. We thus established an AKR-dependent whole-cell biotransformation process that can be used for production of human AKR metabolites on a large scale.

Aldo-keto reductases (AKR) from the AKR1C subfamily catalyze the carbonyl reduction of the novel anticancer drug oracin in man.

In many cases, cancer chemotherapy still obtains unsatisfactory response rates, rare complete remissions and responses of relatively short duration. Therefore, more effective drugs with new structures against cancer are continuously sought. Oracin, 6-[2-(2-hydroxyethyl)-aminoethyl]-5,11-dioxo-5,6-dihydro-11H-indeno[1,2-c]isoquinoline, is a new anticancer drug which is presently in phase II clinical trials. Pharmacokinetic studies have revealed that oracin undergoes metabolic inactivation by carbonyl reduction. Since metabolic inactivation contributes to chemotherapy resistance, detailed knowledge about the participating enzymes is necessary. In the present study, we identified three members of the aldo-keto reductase (AKR) superfamily to mediate oracin carbonyl reduction in man. For AKR1C1, 1C2 and 1C4, purified from human liver cytosol, we could determine the kinetics and catalytic efficiencies. In addition, we investigated the stereospecificity of formation of reduced oracin (DHO). Whereas AKR1C2 and 1C4 are exclusively (100%) stereospecific for (+)-DHO formation, some 3% of (-)-DHO formation was found for AKR1C1. On the other hand, the activity of AKR1C1 in overall oracin reduction was one order of magnitude higher compared to AKR1C2 and 1C4. Detailed knowledge about all enzymes involved in oracin detoxification may help to improve an anticancer regimen by co-application of respective inhibitors.

Rat NAD+-dependent 3alpha-hydroxysteroid dehydrogenase (AKR1C17): a member of the aldo-keto reductase family highly expressed in kidney cytosol.

Mammalian 3alpha-hydroxysteroid dehydrogenases (3alpha-HSDs) have been divided into two types: Cytosolic NADP(H)-dependent 3alpha-HSDs belonging to the aldo-keto reductase family, and mitochondrial and microsomal NAD(+)-dependent 3alpha-HSDs belonging to the short-chain dehydrogenase/reductase family. In this study, we characterized a rat aldo-keto reductase (AKR1C17), whose functions are unknown. The recombinant AKR1C17 efficiently oxidized 3alpha-hydroxysteroids and bile acids using NAD(+) as the preferred coenzyme at an optimal pH of 7.4-9.5, and was inhibited by ketamine and organic anions. The mRNA for AKR1C17 was detected specifically in rat kidney, where the enzyme was more highly expressed as a cytosolic protein than NADP(H)-dependent 3alpha-HSD (AKR1C9). Thus, AKR1C17 represents a novel NAD(+)-dependent type of cytosolic 3alpha-HSD with unique inhibitor sensitivity and tissue distribution. In addition, the replacement of Gln270 and Glu276 of AKR1C17 with the corresponding residues of NADP(H)-dependent 3alpha-HSD resulted in a switch in favor of NADP(+) specificity, suggesting their key roles in coenzyme specificity.

Cloning and expression of chicken 20-hydroxysteroid dehydrogenase.

The ligand specificity and activation of steroid receptors depend considerably on the enzymatic activities involved in local pre-receptor synthesis and the metabolism of the steroids. Several enzymes in particular, steroid dehydrogenases have been shown to participate in this process. Here we report the isolation of 20-hydroxysteroid dehydrogenase (ch20HSD) cDNA from chicken intestine and the distribution of ch20HSD mRNA and 20-reductase activity in various avian tissues. Using a reverse transcription PCR and comparison with the known sequences of mammalian 20betaHSDs, we have isolated a new ch20HSD cDNA. This cDNA predicted 276 amino acid residues that shared about 75% homology with mammalian 20betaHSD. Sequences specific to the short-chain dehydrogenase/reductase superfamily (SDR) were found, the Gly-X-X-X-Gly-X-Gly cofactor-binding motif (residues 11-17) and the catalytic activity motif Tyr-X-X-X-Lys (residues 193-197). The cDNA coding for ch20HSD was expressed in Escherichia coli by placing it under isopropylthiogalactoside (IPTG) inducible control. Both the IPTG cells of E. coli and the isolated recombinant protein reduced progesterone to 20-dihydroprogesterone, corticosterone to 20-dihydrocorticosterone and 5alpha-dihydrotestosterone to its 3-ol derivative. The 20-reductase and 3-reductase activities of ch20HSD catalyzed both 3alpha/beta- and 20alpha/20beta-epimers. The mRNA transcripts of ch20HSD were found in the kidney, colon, and testes; weaker expression was also found in the heart, ovaries, oviduct, brain, liver, and ileum. 20-Reductase activity has been proven in tissue slices of kidney, colon, ileum, liver, oviduct, testis, and ovary; whereas the activity was nearly absent in the heart and brain. A similar distribution of 20-reductase activity was found in tissue homogenates measured under V(max) conditions. These results suggest that chicken 20HSD is the latest member of the SDR superfamily to be found, is expressed in many avian tissues and whose precise role remains to be determined.

Molecular docking simulations of steroid substrates into human cytosolic hydroxysteroid dehydrogenases (AKR1C1 and AKR1C2): insights into positional and stereochemical preferences.

AKR1C1 and AKR1C2 are human cytosolic hydroxysteroid dehydrogenases, which play pivotal roles in the metabolism and action of natural and synthetic steroid hormones. The two enzymes are highly homologous, and have distinct positional and stereochemical preferences with various substrates. We performed molecular docking simulations of three steroid substrates, including an androgen (5alpha-dihydrotestosterone, DHT), a progestin (progesterone, PRO), and a synthetic hormone ([7alpha,17alpha]-17-hydroxy-7-methyl-19-norpregn-5(10)-en-20-yn-3-one or tibolone, TIB), into the active sites of the two enzymes. For each substrate and enzyme pair, the activity inferred by the "productive" docking models (in which the spatial arrangement of the steroid and the cofactor would permit a reaction) matched the experimentally observed positional and stereochemical outcome. These productive conformations were energetically and statistically favored except for TIB and PRO with AKR1C2, where experimentally strong substrate inhibition and low activity were observed, respectively. Results showed that (i) a 3-ketosteroid (DHT) and a 20-ketosteroid (PRO) were reduced by AKR1C1 since the carbonyl groups could occupy the same position by "backwards" binding of steroids; (ii) 3alpha-reduced (DHT) and 3beta-reduced (TIB) products were formed by AKR1C2 since the angular methyl groups of the steroids were inverted by "upside-down" binding of steroids; and (iii) the 3beta- and 3alpha-reduction of DHT by AKR1C1 and AKR1C2, respectively occurred since the steroids employed a "swinging" motion to present opposite faces to the cofactor. Favorable nonproductive modes were observed with all substrates in both enzymes in which the steroid was bound at a "near-entry" position and/or an "in-middle" position, which may influence the reaction coordinate.

3alpha/beta,20beta-hydroxysteroid dehydrogenase (porcine testicular carbonyl reductase) also has a cysteine residue that is involved in binding of cofactor NADPH.

Besides residue of the catalytic triad that is conserved in the short-chain dehydrogenase/reductase (SDR) superfamily, a Cys side chain reportedly plays functional roles in NADP-dependent 15-hydroxyprostaglandin dehydrogenase and human carbonyl reductase (CR). The three-dimensional structure of porcine 3alpha/beta,20beta-hydroxysteroid dehydrogenase, also known as porcine testicular carbonyl reductase, demonstrates the proximity of the Cys 226 side chain to the bound NADP. However, no clear explanation with respect to the basis of the catalytic function of the Cys residue is yet available. By chemical modification, point mutation, and kinetic analysis, we determine that two Cys residues, Cys 149 and Cys 226, are involved in the enzyme activity. Furthermore, we found that pretreatment with NADP markedly protects the enzyme from inactivation by 4-(hydroxyl mercury) benzoic acid (4-HMB), thereby confirming that Cys 226 is involved in binding of the cofactor. On the basis of the tertiary structure of 3alpha/beta,20beta-HSD, the possible roles of Cys residues, especially that of Cys 226, in enzyme action and in the binding of cofactor NADPH are discussed.