5?-Reduced steroids and human ?(4)-3-ketosteroid 5?-reductase (AKR1D1).
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ABSTRACT:
SUBMITTER: Chen M
PROVIDER: S-EPMC3971473 | biostudies-literature | 2014 May
REPOSITORIES: biostudies-literature
Publications
5β-Reduced steroids and human Δ(4)-3-ketosteroid 5β-reductase (AKR1D1).
Steroids 20140208
5β-Reduced steroids are non-planar steroids that have a 90° bend in their structure to create an A/B cis-ring junction. This novel property is required for bile-acids to act as emulsifiers, but in addition 5β-reduced steroids have remarkable physiology and may act as potent tocolytic agents, endogenous cardiac glycosides, neurosteroids, and can act as ligands for orphan and membrane bound receptors. In humans there is only a single 5β-reductase gene AKR1D1, which encodes Δ(4)-3-ketosteroid-5β-re ...[more]
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Project description:Human steroid 5β-reductase (aldo-keto reductase 1D1) catalyzes the stereospecific NADPH-dependent reduction of the C4-C5 double bond of Δ(4)-ketosteroids to yield an A/B cis-ring junction. This cis-configuration is crucial for bile acid biosynthesis and plays important roles in steroid metabolism. The biochemical properties of the enzyme have not been thoroughly studied and conflicting data have been reported, partially due to the lack of highly homogeneous protein. In the present study, we systematically determined the substrate specificity of homogeneous human recombinant AKR1D1 using C18, C19, C21, and C27 Δ(4)-ketosteroids and assessed the pH-rate dependence of the enzyme. Our results show that AKR1D1 proficiently reduced all the steroids tested at physiological pH, indicating AKR1D1 is the only enzyme necessary for all the 5β-steroid metabolites present in humans. Substrate inhibition was observed with C18 to C21 steroids provided that the C11 position was unsubstituted. This structure activity relationship can be explained by the existence of a small alternative substrate binding pocket revealed by the AKR1D1 crystal structure. Non-steroidal anti-inflammatory drugs which are potent inhibitors of the related AKR1C enzymes do not inhibit AKR1D1. By contrast chenodeoxycholate and ursodeoxycholate were found to be potent non-competitive inhibitors suggesting that bile-acids may regulate their own synthesis at the level of AKR1D1 inhibition.
| S-EPMC3056882 | biostudies-literature
Project description:Steroid hormones, including glucocorticoids and androgens, exert a wide variety of effects in the body across almost all tissues. The steroid A-ring 5β-reductase (AKR1D1) is expressed in human liver and testes, and three splice variants have been identified (AKR1D1-001, AKR1D1-002, AKR1D1-006). Amongst these, AKR1D1-002 is the best described; it modulates steroid hormone availability and catalyses an important step in bile acid biosynthesis. However, specific activity and expression of AKR1D1-001 and AKR1D1-006 are unknown. Expression of AKR1D1 variants were measured in human liver biopsies and hepatoma cell lines by qPCR. Their three-dimensional (3D) structures were predicted using in silico approaches. AKR1D1 variants were overexpressed in HEK293 cells, and successful overexpression confirmed by qPCR and Western blotting. Cells were treated with either cortisol, dexamethasone, prednisolone, testosterone or androstenedione, and steroid hormone clearance was measured by mass spectrometry. Glucocorticoid and androgen receptor activation were determined by luciferase reporter assays. AKR1D1-002 and AKR1D1-001 are expressed in human liver, and only AKR1D1-006 is expressed in human testes. Following overexpression, AKR1D1-001 and AKR1D1-006 protein levels were lower than AKR1D1-002, but significantly increased following treatment with the proteasomal inhibitor, MG-132. AKR1D1-002 efficiently metabolised glucocorticoids and androgens and decreased receptor activation. AKR1D1-001 and AKR1D1-006 poorly metabolised dexamethasone, but neither protein metabolised cortisol, prednisolone, testosterone or androstenedione. We have demonstrated the differential expression and role of AKR1D1 variants in steroid hormone clearance and receptor activation in vitro. AKR1D1-002 is the predominant functional protein in steroidogenic and metabolic tissues. In addition, AKR1D1-001 and AKR1D1-006 may have a limited, steroid-specific role in the regulation of dexamethasone action.
| S-EPMC7965358 | biostudies-literature
Project description:AKR1D1 (steroid 5beta-reductase) reduces all Delta(4)-3-ketosteroids to form 5beta-dihydrosteroids, a first step in the clearance of steroid hormones and an essential step in the synthesis of all bile acids. The reduction of the carbon-carbon double bond in an alpha,beta-unsaturated ketone by 5beta-reductase is a unique reaction in steroid enzymology because hydride transfer from NADPH to the beta-face of a Delta(4)-3-ketosteroid yields a cis-A/B-ring configuration with an approximately 90 degrees bend in steroid structure. Here, we report the first x-ray crystal structure of a mammalian steroid hormone carbon-carbon double bond reductase, human Delta(4)-3-ketosteroid 5beta-reductase (AKR1D1), and its complexes with intact substrates. We have determined the structures of AKR1D1 complexes with NADP(+) at 1.79- and 1.35-A resolution (HEPES bound in the active site), NADP(+) and cortisone at 1.90-A resolution, NADP(+) and progesterone at 2.03-A resolution, and NADP(+) and testosterone at 1.62-A resolution. Complexes with cortisone and progesterone reveal productive substrate binding orientations based on the proximity of each steroid carbon-carbon double bond to the re-face of the nicotinamide ring of NADP(+). This orientation would permit 4-pro-(R)-hydride transfer from NADPH. Each steroid carbonyl accepts hydrogen bonds from catalytic residues Tyr(58) and Glu(120). The Y58F and E120A mutants are devoid of activity, supporting a role for this dyad in the catalytic mechanism. Intriguingly, testosterone binds nonproductively, thereby rationalizing the substrate inhibition observed with this particular steroid. The locations of disease-linked mutations thought to be responsible for bile acid deficiency are also revealed.
| S-EPMC2423251 | biostudies-literature
Project description:Human aldo-keto reductase 1D1 (AKR1D1) and AKR1C enzymes are essential for bile acid biosynthesis and steroid hormone metabolism. AKR1D1 catalyzes the 5β-reduction of Δ(4)-3-ketosteroids, whereas AKR1C enzymes are hydroxysteroid dehydrogenases (HSDs). These enzymes share high sequence identity and catalyze 4-pro-(R)-hydride transfer from NADPH to an electrophilic carbon but differ in that one residue in the conserved AKR catalytic tetrad, His(120) (AKR1D1 numbering), is substituted by a glutamate in AKR1D1. We find that the AKR1D1 E120H mutant abolishes 5β-reductase activity and introduces HSD activity. However, the E120H mutant unexpectedly favors dihydrosteroids with the 5α-configuration and, unlike most of the AKR1C enzymes, shows a dominant stereochemical preference to act as a 3β-HSD as opposed to a 3α-HSD. The catalytic efficiency achieved for 3β-HSD activity is higher than that observed for any AKR to date. High resolution crystal structures of the E120H mutant in complex with epiandrosterone, 5β-dihydrotestosterone, and Δ(4)-androstene-3,17-dione elucidated the structural basis for this functional change. The glutamate-histidine substitution prevents a 3-ketosteroid from penetrating the active site so that hydride transfer is directed toward the C3 carbonyl group rather than the Δ(4)-double bond and confers 3β-HSD activity on the 5β-reductase. Structures indicate that stereospecificity of HSD activity is achieved because the steroid flips over to present its α-face to the A-face of NADPH. This is in contrast to the AKR1C enzymes, which can invert stereochemistry when the steroid swings across the binding pocket. These studies show how a single point mutation in AKR1D1 can introduce HSD activity with unexpected configurational and stereochemical preference.
| S-EPMC3351325 | biostudies-literature
Project description:Human steroid-5β-reductase (aldo-keto reductase 1D1, AKR1D1) stereospecifically reduces Δ(4)-3-ketosteroids to 5β-dihydrosteroids and is essential for steroid hormone metabolism and bile acid biosynthesis. Genetic defects in AKR1D1 cause bile acid deficiency that leads to life threatening neonatal hepatitis and cholestasis. The disease-associated P133R mutation caused significant decreases in catalytic efficiency with both the representative steroid (cortisone) and the bile acid precursor (7α-hydroxycholest-4-en-3-one) substrates. Pro133 is a second shell residue to the steroid binding channel and is distal to both the cofactor binding site and the catalytic center. Strikingly, the P133R mutation caused over a 40-fold increase in Kd values for the NADP(H) cofactors and increased the rate of release of NADP(+) from the enzyme by 2 orders of magnitude when compared to the wild type enzyme. By contrast the effect of the mutation on Kd values for steroids were 10-fold or less. The reduced affinity for the cofactor suggests that the mutant exists largely in the less stable cofactor-free form in the cell. Using stopped-flow spectroscopy, a significant reduction in the rate of the chemical step was observed in multiple turnover reactions catalyzed by the P133R mutant, possibly due to the altered position of NADPH. Thus, impaired NADPH binding and hydride transfer is the molecular basis for bile acid deficiency in patients with the P133R mutation. Results revealed that optimal cofactor binding is vulnerable to distant structural perturbation, which may apply to other disease-associated mutations in AKR1D1, all of which occur at conserved residues and are unstable.
| S-EPMC4696769 | biostudies-literature
Project description:Active sex hormones such as testosterone and progesterone are metabolized to tetrahydrosteroids in the liver to terminate hormone action. One main metabolic pathway, the 5?-pathway, involves 5?-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 5?-dihydrotestosterone, 5?-pregnane-3,20-dione and 20?-hydroxy-5?-pregnan-3-one, the intermediate 5?-dihydrosteroids on the 5?-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 3?-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 5?-tetrahydrosteroid of testosterone, but indicated differential routes and roles of human AKR1C for the hepatic formation of 5?-tetrahydrosteroids of progesterone. Comparison of the kinetics of the AKR1C1-AKR1C4-catalysed reactions with those of AKR1D1 suggested that the three intermediate 5?-dihydrosteroids derived from testosterone and progesterone are unlikely to accumulate in liver, and that the identities and levels of 5?-reduced metabolites formed in peripheral tissues will be governed by the local expression of AKR1D1 and AKR1C1-AKR1C3.
| S-EPMC3825703 | biostudies-literature
Project description:Human steroid 5beta-reductase (aldo-keto reductase (AKR) 1D1) catalyzes reduction of Delta(4)-ene double bonds in steroid hormones and bile acid precursors. We have reported the structures of an AKR1D1-NADP(+) binary complex, and AKR1D1-NADP(+)-cortisone, AKR1D1-NADP(+)-progesterone and AKR1D1-NADP(+)-testosterone ternary complexes at high resolutions. Recently, structures of AKR1D1-NADP(+)-5beta-dihydroprogesterone complexes showed that the product is bound unproductively. Two quite different mechanisms of steroid double bond reduction have since been proposed. However, site-directed mutagenesis supports only one mechanism. In this mechanism, the 4-pro-R hydride is transferred from the re-face of the nicotinamide ring to C5 of the steroid substrate. E120, a unique substitution in the AKR catalytic tetrad, permits a deeper penetration of the steroid substrate into the active site to promote optimal reactant positioning. It participates with Y58 to create a "superacidic" oxyanion hole for polarization of the C3 ketone. A role for K87 in the proton relay proposed using the AKR1D1-NADP(+)-5beta-dihydroprogesterone structure is not supported.
| S-EPMC2675190 | biostudies-literature
Project description:Background3-Ketosteroid Δ1-dehydrogenases (KSTDs) are the enzymes involved in microbial cholesterol degradation and modification of steroids. They catalyze dehydrogenation between C1 and C2 atoms in ring A of the polycyclic structure of 3-ketosteroids. KSTDs substrate spectrum is broad, even though most of them prefer steroids with small substituents at the C17 atom. The investigation of the KSTD's substrate specificity is hindered by the poor solubility of the hydrophobic steroids in aqueous solutions. In this paper, we used 2-hydroxpropyl-β-cyclodextrin (HBC) as a solubilizing agent in a study of the KSTDs steady-state kinetics and demonstrated that substrate bioavailability has a pivotal impact on enzyme specificity.ResultsMolecular dynamics simulations on KSTD1 from Rhodococcus erythropolis indicated no difference in ΔGbind between the native substrate, androst-4-en-3,17-dione (AD; - 8.02 kcal/mol), and more complex steroids such as cholest-4-en-3-one (- 8.40 kcal/mol) or diosgenone (- 6.17 kcal/mol). No structural obstacle for binding of the extended substrates was also observed. Following this observation, our kinetic studies conducted in the presence of HBC confirmed KSTD1 activity towards both types of steroids. We have compared the substrate specificity of KSTD1 to the other enzyme known for its activity with cholest-4-en-3-one, KSTD from Sterolibacterium denitrificans (AcmB). The addition of solubilizing agent caused AcmB to exhibit a higher affinity to cholest-4-en-3-one (Ping-Pong bi bi KmA = 23.7 μM) than to AD (KmA = 529.2 μM), a supposedly native substrate of the enzyme. Moreover, we have isolated AcmB isoenzyme (AcmB2) and showed that conversion of AD and cholest-4-en-3-one proceeds at a similar rate. We demonstrated also that the apparent specificity constant of AcmB for cholest-4-en-3-one (kcat/KmA = 9.25∙106 M-1 s-1) is almost 20 times higher than measured for KSTD1 (kcat/KmA = 4.71∙105 M-1 s-1).ConclusionsWe confirmed the existence of AcmB preference for a substrate with an undegraded isooctyl chain. However, we showed that KSTD1 which was reported to be inactive with such substrates can catalyze the reaction if the solubility problem is addressed.
| S-EPMC8220720 | biostudies-literature
Project description:Mammalian steroid 5β-reductases belong to the Aldo-Keto Reductase 1D sub-family and are essential for the formation of A-ring 5β-reduced steroids. Steroid 5β-reduction is required for the biosynthesis of bile-acids and the metabolism of all steroid hormones that contain a Δ4-3-ketosteroid functionally to yield the 5β-reduced metabolites. In mammalian AKR1D enzymes the conserved catalytic tetrad found in all AKRs (Y55, H117, K84 and D50) has changed in that the conserved H117 is replaced with a glutamic acid (E120). E120 may act as a "superacid" to facilitate enolization of the Δ4-ketosteroid. In addition, the absence of the bulky imidazole side chain of histidine in E120 permits the steroid to penetrate deeper into the active site so that hydride transfer can occur to the steroid C5 position. In murine steroid 5β-reductase AKR1D4, we find that there is a long-form, with an 18 amino-acid extension at the N-terminus (AKR1D4L) and a short-form (AKR1D4S), where the latter is recognized as AKR1D4 by the major data-bases. Both enzymes were purified to homogeneity and product profiling was performed. With progesterone and cortisol, AKR1D4L and AKR1D4S catalyzed smooth conversion to the 5β-dihydrosteroids. However, with Δ4-androstene-3,17-dione as substrate, a mixture of products was observed which included, 5β-androstane-3,17-dione (expected) but 3α-hydroxy-5β- androstan-17-one was also formed. The latter compound was distinguished from its isomeric 3β-hydroxy-5β-androstan-17-one by forming picolinic acid derivatives followed by LC-MS. These data show that AKR1D4L and AKR1D4S also act as 3α-hydroxysteroid dehydrogenases when presented with Δ4-androstene-3,17-dione and suggest that E120 alters the position the steroid to enable a correct trajectory for hydride transfer and may not act as a "superacid".
| S-EPMC6500757 | biostudies-literature
Project description:BackgroundA substantial group of patients with cholestatic liver disease in infancy excrete, as the major urinary bile acids, the glycine and taurine conjugates of 7alpha-hydroxy-3-oxo-4-cholenoic acid and 7alpha,12alpha-dihydroxy-3-oxo-4-cholenoic acid. It has been proposed that some (but not all) of these have mutations in the gene encoding delta(4)-3-oxosteroid 5beta-reductase (SRD5B1; AKR1D1, OMIM 604741).AimsOur aim was to identify mutations in the SRD5B1 gene in patients in whom chenodeoxycholic acid and cholic acid were absent or present at low concentrations in plasma and urine, as these seemed strong candidates for genetic 5beta-reductase deficiency.Patients and subjectsWe studied three patients with neonatal onset cholestatic liver disease and normal gamma-glutamyl transpeptidase in whom 3-oxo-delta(4) bile acids were the major bile acids in urine and plasma and saturated bile acids were at low concentration or undetectable. Any base changes detected in SRD5B1 were sought in the parents and siblings and in 50 ethnically matched control subjects.MethodsDNA was extracted from blood and the nine exons of SRD5B1 were amplified and sequenced. Restriction enzymes were used to screen the DNA of parents, siblings, and controls.ResultsMutations in the SRD5B1 gene were identified in all three children. Patient MS was homozygous for a missense mutation (662 C>T) causing a Pro198Leu amino acid substitution; patient BH was homozygous for a single base deletion (511 delT) causing a frame shift and a premature stop codon in exon 5; and patient RM was homozygous for a missense mutation (385 C>T) causing a Leu106Phe amino acid substitution. All had liver biopsies showing a giant cell hepatitis; in two, prominent extramedullary haemopoiesis was noted. MS was cured by treatment with chenodeoxycholic acid and cholic acid; BH showed initial improvement but then deteriorated and required liver transplantation; RM had advanced liver disease when treatment was started and also progressed to liver failure.ConclusionsAnalysis of blood samples for SRD5B1 mutations can be used to diagnose genetic 5beta-reductase deficiency and distinguish these patients from those who have another cause of 3-oxo-delta(4) bile aciduria, for example, severe liver damage. Patients with genetic 5beta-reductase deficiency may respond well to treatment with chenodeoxycholic acid and cholic acid if liver disease is not too advanced.
| S-EPMC1773813 | biostudies-literature