Steroid Substrate Specificity Research Articles

Enzymatic synthesis of a skin active ingredient - glochidone by 3-ketosteroid dehydrogenase from Sterolibacterium denitrificans

In this study, we applied AcmB2, sourced from Sterolibacterium denitrificans, to catalyze the oxidative dehydrogenation of 3-ketolupeol (lupenone), a derivative of lupeol, triterpene obtained from birch bark. This enzymatic Δ1-dehydrogenation catalyzed by AcmB2 yielded glochidone, a bioactive compound frequently obtained from medicinal plants like Salvia trichoclada and Maytenus boria. Glochidone is known for its broad biological activities, including antibacterial, antifungal, anti-inflammatory, anticancer, antidiabetic as well as acetylcholinesterase inhibition. Our research demonstrates >99% conversion efficiency with 100% regioselectivity of the reaction. The effective conversion to glochidone employed an electron acceptor e.g., potassium hexacyanoferrate III, in mild, environmentally friendly conditions: 8–16% 2-hydroxypropyl-β-cyclodextrin, and 2–3% 2-methoxyethanol. AcmB2 reaction optimum was determined at pH 8.0 and 30 °C. Enzyme's biochemical attributes such as electron acceptor type, concentration and steroid substrate specificity were investigated. Among 4-, 5- and 6-ring steroid derivatives androst-4-en-3,17-dione and testosterone propionate were determined as the best substrates of AcmB2. Δ1-Dehydrogenation of substrates such as lupenone, diosgenone and 3-ketopetromyzonol was confirmed. We have assessed the antioxidant and rejuvenating characteristics of glochidone as an active component in formulations, considering its precursors, lupeol, and lupenone as well. Glochidone exhibited limited antioxidant and chelating capabilities compared to lupeol and reference compounds. However, it demonstrated robust rejuvenating properties, with a sirtuin induction level of 61.5 ± 1.87%, notably surpassing that of the reference substance, E-resveratrol (45.15 ± 0.09%). Additionally, glochidone displayed 26.5±0.67 and 19.41±0.76% inhibition of elastase and collagenase, respectively. The safety of all studied triterpenes was confirmed on skin reconstructed human Epidermis model. These findings provide valuable insights into the potential applications of glochidone in formulations aimed at addressing skin health concerns. This research presents the first example of an enzyme in the 3-ketosteroid dehydrogenase (KstD) family catalyzing the Δ1-dehydrogenation of a pentacyclic triterpene. We also explored structural differences between AcmB, AcmB2, and related KstDs pointing to G52 and P532 as potentially responsible for the unique substrate specificity of AcmB2. Our findings not only highlight the enzyme's capabilities but also present novel enzymatic pathways for bioactive compound synthesis.

Rhodococcus rhodochrous DSM 43269 3-Ketosteroid 9α-Hydroxylase, a Two-Component Iron-Sulfur-Containing Monooxygenase with Subtle Steroid Substrate Specificity

This paper reports the biochemical characterization of a purified and reconstituted two-component 3-ketosteroid 9alpha-hydroxylase (KSH). KSH of Rhodococcus rhodochrous DSM 43269, consisting of a ferredoxin reductase (KshB) and a terminal oxygenase (KshA), was heterologously expressed in Escherichia coli. E. coli cell cultures, expressing both KshA and KshB, converted 4-androstene-3,17-dione (AD) into 9alpha-hydroxy-4-AD (9OHAD) with a >60% molar yield over 48 h of incubation. Coexpression and copurification were critical to successfully obtain pure and active KSH. Biochemical analysis revealed that the flavoprotein KshB is an NADH-dependent reductase using flavin adenine dinucleotide as a cofactor. Reconstitution experiments confirmed that KshA, KshB, and NADH are essential for KSH activity with steroid substrates. KSH hydroxylation activity was inhibited by several divalent metal ions, especially by zinc. The reconstituted KSH displayed subtle steroid substrate specificity; a range of 3-ketosteroids, i.e., 5alpha-Eta, 5beta-Eta, Delta1, and Delta4 steroids, could act as KSH substrates, provided that they had a short side chain. The formation of 9OHAD from AD by KSH was confirmed by liquid chromatography-mass spectrometry analysis and by the specific enzymatic conversion of 9OHAD into 3-hydroxy-9,10-secoandrost-1,3,5(10)-triene-9,17-dione using 3-ketosteroid Delta1-dehydrogenase. Only a single KSH is encoded in the genome of the human pathogen Mycobacterium tuberculosis H37Rv, shown to be important for survival in macrophages. Since no human KSH homolog exists, the M. tuberculosis enzyme may provide a novel target for treatment of tuberculosis. Detailed knowledge about the biochemical properties of KSH thus is highly relevant in the research fields of biotechnology and medicine.

Structure and function of human 17β-hydroxysteroid dehydrogenases

17β-Hydroxysteroid dehydrogenases (17β-HSDs) catalyze the NAD(P)(H) dependent oxidoreduction at C17 oxo/β-hydroxyl groups of androgen and estrogen hormones. This reversible reaction constitutes an important pre-receptor control mechanism for nuclear receptor ligands, since the conversion “switches” between the 17β–OH receptor ligands and their inactive 17-oxo metabolites. At present, 14 mammalian 17β-HSDs are described, of which at least 11 exist within the human genome, encoded by different genes. The enzymes differ in their expression pattern, nucleotide cofactor preference, steroid substrate specificity and subcellular localization, and thus constitute a complex system ensuring cell-specific adaptation and regulation of sex steroid hormone levels. Broad and overlapping substrate specificities with enzymes involved in lipid metabolism suggest interactions of several 17β-HSDs with other metabolic pathways. Several 17β-HSDs enzymes constitute promising drug targets, of particular importance in cancer, metabolic diseases, neurodegeneration and possibly immunity.

Human 3α-hydroxysteroid dehydrogenase isoforms (AKR1C1–AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones

The kinetic parameters, steroid substrate specificity and identities of reaction products were determined for four homogeneous recombinant human 3α-hydroxysteroid dehydrogenase (3α-HSD) isoforms of the aldo-keto reductase (AKR) superfamily. The enzymes correspond to type 1 3α-HSD (AKR1C4), type 2 3α(17β)-HSD (AKR1C3), type 3 3α-HSD (AKR1C2) and 20α(3α)-HSD (AKR1C1), and share at least 84% amino acid sequence identity. All enzymes acted as NAD(P)(H)-dependent 3-, 17- and 20-ketosteroid reductases and as 3α-, 17β- and 20α-hydroxysteroid oxidases. The functional plasticity of these isoforms highlights their ability to modulate the levels of active androgens, oestrogens and progestins. Salient features were that AKR1C4 was the most catalytically efficient, with kcat/Km values for substrates that exceeded those obtained with other isoforms by 10–30-fold. In the reduction direction, all isoforms inactivated 5α-dihydrotestosterone (17β-hydroxy-5α-androstan-3-one; 5α-DHT) to yield 5α-androstane-3α,17β-diol (3α-androstanediol). However, only AKR1C3 reduced ∆4-androstene-3,17-dione to produce significant amounts of testosterone. All isoforms reduced oestrone to 17β-oestradiol, and progesterone to 20α-hydroxy-pregn-4-ene-3,20-dione (20α-hydroxyprogesterone). In the oxidation direction, only AKR1C2 converted 3α-androstanediol to the active hormone 5α-DHT. AKR1C3 and AKR1C4 oxidized testosterone to ∆4-androstene-3,17-dione. All isoforms oxidized 17β-oestradiol to oestrone, and 20α-hydroxyprogesterone to progesterone. Discrete tissue distribution of these AKR1C enzymes was observed using isoform-specific reverse transcriptase-PCR. AKR1C4 was virtually liver-specific and its high kcat/Km allows this enzyme to form 5α/5β-tetrahydrosteroids robustly. AKR1C3 was most prominent in the prostate and mammary glands. The ability of AKR1C3 to interconvert testosterone with ∆4-androstene-3,17-dione, but to inactivate 5α-DHT, is consistent with this enzyme eliminating active androgens from the prostate. In the mammary gland, AKR1C3 will convert ∆4-androstene-3,17-dione to testosterone (a substrate aromatizable to 17β-oestradiol), oestrone to 17β-oestradiol, and progesterone to 20α-hydroxyprogesterone, and this concerted reductive activity may yield a pro-oesterogenic state. AKR1C3 is also the dominant form in the uterus and is responsible for the synthesis of 3α-androstanediol which has been implicated as a parturition hormone. The major isoforms in the brain, capable of synthesizing anxiolytic steroids, are AKR1C1 and AKR1C2. These studies are in stark contrast with those in rat where only a single AKR with positional- and stereo-specificity for 3α-hydroxysteroids exists.

Human 3alpha-hydroxysteroid dehydrogenase isoforms (AKR1C1-AKR1C4) of the aldo-keto reductase superfamily: functional plasticity and tissue distribution reveals roles in the inactivation and formation of male and female sex hormones.

The kinetic parameters, steroid substrate specificity and identities of reaction products were determined for four homogeneous recombinant human 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD) isoforms of the aldo-keto reductase (AKR) superfamily. The enzymes correspond to type 1 3alpha-HSD (AKR1C4), type 2 3alpha(17beta)-HSD (AKR1C3), type 3 3alpha-HSD (AKR1C2) and 20alpha(3alpha)-HSD (AKR1C1), and share at least 84% amino acid sequence identity. All enzymes acted as NAD(P)(H)-dependent 3-, 17- and 20-ketosteroid reductases and as 3alpha-, 17beta- and 20alpha-hydroxysteroid oxidases. The functional plasticity of these isoforms highlights their ability to modulate the levels of active androgens, oestrogens and progestins. Salient features were that AKR1C4 was the most catalytically efficient, with k(cat)/K(m) values for substrates that exceeded those obtained with other isoforms by 10-30-fold. In the reduction direction, all isoforms inactivated 5alpha-dihydrotestosterone (17beta-hydroxy-5alpha-androstan-3-one; 5alpha-DHT) to yield 5alpha-androstane-3alpha,17beta-diol (3alpha-androstanediol). However, only AKR1C3 reduced Delta(4)-androstene-3,17-dione to produce significant amounts of testosterone. All isoforms reduced oestrone to 17beta-oestradiol, and progesterone to 20alpha-hydroxy-pregn-4-ene-3,20-dione (20alpha-hydroxyprogesterone). In the oxidation direction, only AKR1C2 converted 3alpha-androstanediol to the active hormone 5alpha-DHT. AKR1C3 and AKR1C4 oxidized testosterone to Delta(4)-androstene-3,17-dione. All isoforms oxidized 17beta-oestradiol to oestrone, and 20alpha-hydroxyprogesterone to progesterone. Discrete tissue distribution of these AKR1C enzymes was observed using isoform-specific reverse transcriptase-PCR. AKR1C4 was virtually liver-specific and its high k(cat)/K(m) allows this enzyme to form 5alpha/5beta-tetrahydrosteroids robustly. AKR1C3 was most prominent in the prostate and mammary glands. The ability of AKR1C3 to interconvert testosterone with Delta(4)-androstene-3,17-dione, but to inactivate 5alpha-DHT, is consistent with this enzyme eliminating active androgens from the prostate. In the mammary gland, AKR1C3 will convert Delta(4)-androstene-3,17-dione to testosterone (a substrate aromatizable to 17beta-oestradiol), oestrone to 17beta-oestradiol, and progesterone to 20alpha-hydroxyprogesterone, and this concerted reductive activity may yield a pro-oesterogenic state. AKR1C3 is also the dominant form in the uterus and is responsible for the synthesis of 3alpha-androstanediol which has been implicated as a parturition hormone. The major isoforms in the brain, capable of synthesizing anxiolytic steroids, are AKR1C1 and AKR1C2. These studies are in stark contrast with those in rat where only a single AKR with positional- and stereo-specificity for 3alpha-hydroxysteroids exists.

Characterization of the purified pituitary cytosolic NADPH: 5α-dihydroprogesterone 3α-hydroxysteroid oxidoreductase

The purified cytosolic 3α-hydroxysteroid oxidoreductase (3α-HSOR) from female rat pituitary which catalyzes the reversible conversion of 5α-dihydroprogesterone (5α-DHP) to 3α,5α-tetrahydroprogesterone (3α,5α-THP) has been characterized in terms of its steroid substrate specificity, dihydrodiol dehydrogenase activity and inhibition by drugs such as medroxyprogesterone and indomethacin. The purified enzyme has a strong preference for the C 21 progestin steroid substrates, 5α-DHP and 3α,5α-THP, over the corresponding C 19 androgenic steroid substrates, 5α-dihydrotestosterone (5α-DHT) and 3α,5α-tetrahydrotestosterone (3α,5α-THT). The apparent K m for 5α-DHP (80 nM) is about 250 times lower than the K m for the androgenic steroid, 5α-DHT (21 μM). In the oxidative direction, the apparent K m for 3α,5α-TP (1.4 μM) is about 3-fold lower than the K m for the androgenic steroid, 3α,5α-THT (4.2 μM). A number of other naturally occurring 3-keto- and 3α(β)-hydroxy-steroids were assessed for their ability to act as inhibitors (alternate substrates) of the 3α-reduction of 5α-DHP catalyzed by the purified 3α-HSOR. None of the 3β- or 5β-isomers had any effect. Of the other 3-keto and 3α- steroids tested, only deoxycorticosterone and the ovarian progestins showed any significant inhibition. These may be acting as inhibitors since there was little, if any, direct 3α-reduction of progesterone to 3α-hydroxy-4-pregnen-20-one. Unlike the liver cytosolic 3α-HSOR, the pituitary enzyme has no associated dihydrodiol (quinone) dehydrogenase activity. This enzyme is similar to other cytosolic 3α-HSORs from liver and brain in that it is potentially inhibited by indomethacin and by medroxyprogesterone.

Functional characterization of two cytochrome P-450s within the mouse, male-specific steroid 16 alpha-hydroxylase gene family: expression in mammalian cells and chimeric proteins.

Two cDNAs, pc16 alpha-2 and pc16 alpha-25, which encode P-450s from within the mouse, male-specific steroid 16 alpha-hydroxylase (C-P-450(16 alpha)) gene family, were transfected into COS-1 cells in order to study catalytic activities of the expressed P-450s. pc16 alpha-2 was shown previously to encode the growth hormone dependent and androgen-dependent C-P-450(16 alpha) in adult male mice (Wong et al., 1987). The sequence of pc16 alpha-25-encoded P-450 (P-450cb) was identical with gene cb within the C-P-450(16 alpha) family. There was 94% and 87% nucleotide and amino acid sequence identity, respectively, between P-450cb and C-P-450(16 alpha). We expressed both P-450s by transfecting their cDNAs into COS-1 cells and found that steroid 16 alpha-hydroxylase activity was catalyzed by C-P-450(16 alpha) but not by P-450cb. In addition to testosterone, progesterone and estradiol were hydroxylated specifically at the 16 alpha-position by the expressed C-P-450(16 alpha). The results indicated that a broad steroid substrate specificity with high regio- and stereoselectivity at that position was a characteristic of C-P-450(16 alpha). We constructed and expressed chimeras between the two P-450s and found that the presence of about two-thirds of the C-P-450(16 alpha) molecule from its C-terminus was necessary for the chimeric cytochrome to maintain steroid 16 alpha-hydroxylase activity.

Characterization and endocrine regulation of the cytochrome P-450 dependent microsomal hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol in the rat ventral prostate.

Of 11 structurally related steroids, 5 alpha-androstane-3 beta,17 beta-diol was the one most efficiently hydroxylated to polar metabolites by microsomes isolated from the rat ventral prostate. The metabolites were identified by gas chromatography-mass spectrometry as 6 alpha-,7 alpha-, and presumably 6 beta-hydroxylated 5 alpha-androstane-3 beta,17 beta-diol. The hydroxylated metabolites were separated and quantitated by high performance liquid chromatography using an aminopropylsilane hypersil column. Maximum velocity (V max) values of 0.36, 0.12, and 0.044 nmol/min X mg of microsomal protein were determined for the 6 alpha-,7 alpha-, and 6 beta-hydroxylase activities, respectively, with an apparent Michaelis-Menten constant (Km) of 30 microM for all hydroxylases. Hydroxylation of 5 alpha-androstane-3 beta,17 beta-diol in prostatic microsomes was NADPH dependent and was inhibited by several cytochrome P-450 inhibitors in vitro and by antibodies against rat liver NADPH-cytochrome P-450 reductase. The enzyme activities were stimulated by treatment of the rats with phenobarbital, beta-naphthoflavone, 5 alpha-androstane-3 beta,17 beta-diol,5 alpha-dihydrotestosterone, and methyltrienolone, whereas the activities were suppressed by ovine PRL, human GH, estradiol benzoate, cholesterol, andrenalectomy , hypophysectomy, and castration. It is concluded that the prostatic 5 alpha-androstane-3 beta,17 beta-diol hydroxylase activities represent a constitutive cytochrome P-450 form in the rat ventral prostate with a high steroid substrate specificity. Endocrine factors are involved in the regulation of the activity of the enzyme. The ratio between the three hydroxylated metabolites was constant in almost all experiments suggesting that only one cytochrome P-450 species was involved in the hydroxylation.

Steroid 5alpha-reductase in cultured human fibroblasts. Biochemical and genetic evidence for two distinct enzyme activities.

Various properties of the steroid 5alpha-reductase have been examined in cell-free extracts of skin and of fibroblasts cultured from genital and nongenital skin from control subjects and from patients with several forms of male pseudohermaphroditism. When 20alpha-hydroxy-4-[1,2-3H] pregnen-3-one was used as substrate, two 5alpha-reductase activities could be demonstrated in intact skin and cultured fibroblasts. The major activity, previously described for microsomes from human prepuce and extracts of cultured foreskin fibroblasts, is characterized by a narrow pH optimum near 5.5 and is limited to fibroblasts derived from genital skin. A second activity, not limited by the site of biopsy, has been demonstrated over a higher and broader range of pH (from 7 to 9); this enzyme activity is found in both genital and nongenital skin and in fibroblasts cultured from all skin regions. Whereas there is wide variability in the activity at pH 5.5 in genital skin fibroblasts, the activity at pH 7 to 9 is similar in fibroblasts derived from all anatomical sites. The two activities exhibit different kinetics with respect to steroid substrate and are also dissimilar in their subcellular distributions. Other properties, such as coenzyme requirement, steroid substrate specificity, and instability with increasing temperature, appear to be similar.