Jens Neunzig

A steroidogenic pathway for sulfonated steroids: the metabolism of pregnenolone sulfate.

In many tissues sulfonated steroids exceed the concentration of free steroids and recently they were also shown to fulfill important physiological functions. While it was previously demonstrated that cholesterol sulfate (CS) is converted by CYP11A1 to pregnenolone sulfate (PregS), further conversion of PregS has not been studied in detail. To investigate whether a steroidogenic pathway for sulfonated steroids exists similar to the one described for free steroids, we examined the interaction of PregS with CYP17A1 in a reconstituted in-vitro system. Difference spectroscopy revealed a Kd-value of 74.8±4.2μM for the CYP17A1-PregS complex, which is 2.5-fold higher compared to the CYP17A1-pregnenolone (Preg) complex. Mass spectrometry experiments proved for the first time that PregS is hydroxylated by CYP17A1 at position C17, identically to pregnenolone. A higher Km- and a lower kcat-value for CYP17A1 using PregS compared with Preg were observed, indicating a 40% reduced catalytic efficiency when using the sulfonated steroid. Furthermore, we analyzed whether the presence of cytochrome b5 (b5) has an influence on the CYP17A1 dependent conversion of PregS, as was demonstrated for Preg. Interestingly, with 17OH-PregS no scission of the 17,20-carbon-carbon bond occurs, when b5 is added to the reconstituted in-vitro system, while b5 promotes the formation of DHEA from 17OH-Preg. When using human SOAT-HEK293 cells expressing CYP17A1 and CPR, we could confirm that PregS is metabolized to 17OH-PregS, strengthening the potential physiological meaning of a pathway for sulfonated steroids.

Dehydroepiandrosterone Sulfate (DHEAS) Stimulates the First Step in the Biosynthesis of Steroid Hormones

Dehydroepiandrosterone sulfate (DHEAS) is the most abundant circulating steroid in human, with the highest concentrations between age 20 and 30, but displaying a significant decrease with age. Many beneficial functions are ascribed to DHEAS. Nevertheless, long-term studies are very scarce concerning the intake of DHEAS over several years, and molecular investigations on DHEAS action are missing so far. In this study, the role of DHEAS on the first and rate-limiting step of steroid hormone biosynthesis was analyzed in a reconstituted in vitro system, consisting of purified CYP11A1, adrenodoxin and adrenodoxin reductase. DHEAS enhances the conversion of cholesterol by 26%. Detailed analyses of the mechanism of DHEAS action revealed increased binding affinity of cholesterol to CYP11A1 and enforced interaction with the electron transfer partner, adrenodoxin. Difference spectroscopy showed K d-values of 40±2.7 µM and 24.8±0.5 µM for CYP11A1 and cholesterol without and with addition of DHEAS, respectively. To determine the K d-value for CYP11A1 and adrenodoxin, surface plasmon resonance measurements were performed, demonstrating a K d-value of 3.0±0.35 nM (with cholesterol) and of 2.4±0.05 nM when cholesterol and DHEAS were added. Kinetic experiments showed a lower Km and a higher kcat value for CYP11A1 in the presence of DHEAS leading to an increase of the catalytic efficiency by 75%. These findings indicate that DHEAS affects steroid hormone biosynthesis on a molecular level resulting in an increased formation of pregnenolone.

Role of steroid sulfatase in steroid homeostasis and characterization of the sulfated steroid pathway: Evidence from steroid sulfatase deficiency

The impact of steroid sulfatase (STS) activity in the circulating levels of both sulfated and unconjugated steroids is only partially known. In addition, the sulfated steroid pathway, a parallel pathway to the one for unconjugated steroids, which uses the same enzymes, has never been characterized in detail before. Patients with steroid sulfatase deficiency (STSD) are unable to enzymatically convert sulfated steroids into their unconjugated forms, and are a good model to elucidate how STS affects steroid biosynthesis and to study the metabolism of sulfated steroids. We quantified unconjugated and sulfated steroids in STSD serum, and compared these results with data obtained from serum of healthy controls. Most sulfated steroids were increased in STSD. However, androstenediol-3-sulfate and epiandrosterone sulfate showed similar levels in both groups, and the concentrations of androsterone sulfate were notably lower. Hydroxylated forms of DHEAS and of pregnenolone sulfate were found to be increased in STSD, suggesting a mechanism to improve the excretion of sulfated steroids. STSD testosterone concentrations were normal, but cholesterol and DHEA were significantly decreased. Additionally, serum bile acids were three-fold higher in STSD. Correlations between concentrations of steroids in each group indicate that 17α-hydroxy-pregnenolone-3-sulfate in men is mainly biosynthesized from the precursor pregnenolone sulfate and androstenediol-3-sulfate from DHEAS. These findings confirm the coexistence of two steroidogenic pathways: one for unconjugated steroids and another one for sulfated steroids. Each pathway is responsible for the synthesis of specific steroids. The equal levels of testosterone, and the reduced level of unconjugated precursors in STSD, support that testosterone is primarily synthesized from sulfated steroids. In consequence, testosterone synthesis in STSD relies on an enzyme with sulfatase activity other than STS. This study reveals that STS is a key player of steroid biosynthesis regulating the availability of circulating cholesterol.

Carboetomidate: an analog of etomidate that interacts weakly with 11β-hydroxylase.

BACKGROUND:Carboetomidate is a pyrrole etomidate analog that is 3 orders of magnitude less potent an inhibitor of in vitro cortisol synthesis than etomidate (an imidazole) and does not inhibit in vivo steroid production. Although carboetomidate’s reduced functional effect on steroid synthesis is thought to reflect lower binding affinity to 11&bgr;-hydroxylase, differential binding to this enzyme has never been experimentally demonstrated. In the current study, we tested the hypothesis that carboetomidate and etomidate bind with differential affinity to 11&bgr;-hydroxylase by comparing their abilities to inhibit photoaffinity labeling of purified enzyme by a photoactivatable etomidate analog and to modify the enzyme’s absorption spectrum in a way that is indicative of ligand binding. In addition, we made a preliminary exploration of the manner in which etomidate and carboetomidate might differentially interact with this site using spectroscopic methods as well as molecular modeling techniques to better understand the structural basis for their selectivity. METHODS:The ability of azi-etomidate to inhibit cortisol synthesis was tested by assessing its ability to inhibit cortisol synthesis by H295R cells. The binding affinities of etomidate and carboetomidate to 11&bgr;-hydroxylase were compared by assessing their abilities to (1) inhibit photoincorporation of the photolabile etomidate analog [3H]azi-etomidate into the enzyme and (2) modify the absorption spectrum of the enzyme’s heme group. In silico docking studies of etomidate, carboetomidate, and azi-etomidate binding to 11&bgr;-hydroxylase were performed using the computer software GOLD. RESULTS:Similar to etomidate, azi-etomidate potently inhibits in vitro cortisol synthesis. Etomidate inhibited [3H]azi-etomidate photolabeling of 11&bgr;-hydroxylase in a concentration-dependent manner. At a concentration of 40 µM, etomidate reduced photoincorporation of [3H]azi-etomidate by 96% ± 1% whereas carboetomidate had no experimentally detectable effect. On addition of etomidate to 11&bgr;-hydroxylase, a type 2 difference spectrum was produced indicative of etomidate complexation with the enzyme’s heme iron; carboetomidate had no effect whereas azi-etomidate produced a reverse type 1 spectrum. Computer modeling studies predicted that etomidate, carboetomidate, and azi-etomidate can fit into the heme-containing pocket that forms 11&bgr;-hydroxylase’s active site and pose with their carbonyl oxygens interacting with the heme iron and their phenyl rings stacking with phenylalanine-80. However, additional unique poses were identified for etomidate and azi-etomidate that likely account for their higher affinities. CONCLUSIONS:Carboetomidate’s reduced ability to suppress in vitro and in vivo steroid synthesis as compared with etomidate reflects its lower binding affinity to 11&bgr;-hydroxylase and may be attributed to carboetomidate’s inability to form a coordination bond with the heme iron located at the enzyme’s active site.

Metabolism of Oral Turinabol by Human Steroid Hormone–Synthesizing Cytochrome P450 Enzymes

The human mitochondrial cytochrome P450 enzymes CYP11A1, CYP11B1, and CYP11B2 are involved in the biosynthesis of steroid hormones. CYP11A1 catalyzes the side-chain cleavage of cholesterol, and CYP11B1 and CYP11B2 catalyze the final steps in the biosynthesis of gluco- and mineralocorticoids, respectively. This study reveals their additional capability to metabolize the xenobiotic steroid oral turinabol (OT; 4-chlor-17β-hydroxy-17α-methylandrosta-1,4-dien-3-on), which is a common doping agent. By contrast, microsomal steroid hydroxylases did not convert OT. Spectroscopic binding assays revealed dissociation constants of 17.7 µM and 5.4 µM for CYP11B1 and CYP11B2, respectively, whereas no observable binding spectra emerged for CYP11A1. Catalytic efficiencies of OT conversion were determined to be 46 min−1 mM−1 for CYP11A1, 741 min−1 mM−1 for CYP11B1, and 3338 min−1 mM−1 for CYP11B2, which is in the same order of magnitude as for the natural substrates but shows a preference of CYP11B2 for OT conversion. Products of OT metabolism by the CYP11B subfamily members were produced at a milligram scale with a recombinant Escherichia coli–based whole-cell system. They were identified by nuclear magnetic resonance spectroscopy to be 11β-OH-OT for both CYP11B isoforms, whereby CYP11B2 additionally formed 11β,18-diOH-OT and 11β-OH-OT-18-al, which rearranges to its tautomeric form 11β,18-expoxy-18-OH-OT. CYP11A1 produces six metabolites, which are proposed to include 2-OH-OT, 16-OH-OT, and 2,16-diOH-OT based on liquid chromatography–tandem mass spectrometry analyses. All three enzymes are shown to be inhibited by OT in their natural function. The extent of inhibition thereby depends on the affinity of the enzyme for OT and the strongest effect was demonstrated for CYP11B2. These findings suggest that steroidogenic cytochrome P450 enzymes can contribute to drug metabolism and should be considered in drug design and toxicity studies.

2β- and 16β-hydroxylase activity of CYP11A1 and direct stimulatory effect of estrogens on pregnenolone formation

The biosynthesis of steroid hormones in vertebrates is initiated by the cytochrome P450 CYP11A1, which performs the side-chain cleavage of cholesterol thereby producing pregnenolone. In this study, we report a direct stimulatory effect of the estrogens estradiol and estrone onto the pregnenolone formation in a reconstituted in vitro system consisting of purified CYP11A1 and its natural redox partners. We demonstrated the formation of new products from 11-deoxycorticosterone (DOC), androstenedione, testosterone and dehydroepiandrosterone (DHEA) during the in vitro reaction catalyzed by CYP11A1. In addition, we also established an Escherichia coli-based whole-cell biocatalytic system consisting of CYP11A1 and its redox partners to obtain sufficient yields of products for NMR-characterization. Our results indicate that CYP11A1, in addition to the previously described 6β-hydroxylase activity, possesses a 2β-hydroxylase activity towards DOC and androstenedione as well as a 16β-hydroxylase activity towards DHEA. We also showed that CYP11A1 is able to perform the 6β-hydroxylation of testosterone, a reaction that has been predominantly attributed to CYP3A4. Our results are the first evidence that sex hormones positively regulate the overall production of steroid hormones suggesting the need to reassess the role of CYP11A1 in steroid hormone biosynthesis and its substrate-dependent mechanistic properties.

The steroid metabolite 16(β)-OH-androstenedione generated by CYP21A2 serves as a substrate for CYP19A1

The 21-hydroxylase (CYP21A2) is a steroidogenic enzyme crucial for the synthesis of mineralo- and glucocorticoids. It is described to convert progesterone as well as 17-OH-progesterone, through a hydroxylation at position C21, into 11-deoxycorticosterone (DOC) and 11-deoxycortisol (RSS), respectively. In this study we unraveled CYP21A2 to have a broader steroid substrate spectrum than assumed. Utilizing a reconstituted in vitro system, consisting of purified human CYP21A2 and human cytochrome P450 reductase (CPR) we demonstrated that CYP21A2 is capable to metabolize DOC, RSS, androstenedione (A4) and testosterone (T). In addition, the conversion of A4 rendered a product whose structure was elucidated through NMR spectroscopy, showing a hydroxylation at position C16-beta. The androgenic properties of this steroid metabolite, 16(β)-OH-androstenedione (16bOHA4), were investigated and compared with A4. Both steroid metabolites were shown to be weak agonists for the human androgen receptor. Moreover, the interaction of 16bOHA4 with the aromatase (CYP19A1) was compared to that of A4, indicating that the C16 hydroxyl group does not influence the binding with CYP19A1. In contrast, the elucidation of the kinetic parameters showed an increased Km and decreased kcat value resulting in a 2-fold decreased catalytic efficiency compared to A4. These findings were in accordance with our docking studies, revealing a similar binding conformation and distance to the heme iron of both steroids. Furthermore, the product of 16bOHA4, presumably 16-hydroxy-estrone (16bOHE1), was investigated with regard to its estrogenic activity, which was negligible compared to estradiol and estrone. Finally, 16bOHA4 was found to be present in a patient with 11-hydroxylase deficiency and in a patient with an endocrine tumor. Taken together, this study provides novel information on the steroid hormone biosynthesis and presents a new method to detect further potential relevant novel steroid metabolites.

A Novel NADPH-dependent flavoprotein reductase from Bacillus megaterium acts as an efficient cytochrome P450 reductase.

Cytochromes P450 (P450s) require electron transfer partners to catalyze substrate conversions. With regard to biotechnological approaches, the elucidation of novel electron transfer proteins is of special interest, as they can influence the enzymatic activity and specificity of the P450s. In the current work we present the identification and characterization of a novel soluble NADPH-dependent diflavin reductase from Bacillus megaterium with activity towards a bacterial (CYP106A1) and a microsomal (CYP21A2) P450 and, therefore, we referred to it as B. megaterium cytochrome P450 reductase (BmCPR). Sequence analysis of the protein revealed besides the conserved FMN-, FAD- and NADPH-binding motifs, the presence of negatively charged cluster, which is thought to represent the interaction domain with P450s and/or cytochrome c. BmCPR was expressed and purified to homogeneity in Escherichia coli. The purified BmCPR exhibited a characteristic diflavin reductase spectrum, and showed a cytochrome c reducing activity. Furthermore, in an in vitro reconstituted system, the BmCPR was able to support the hydroxylation of testosterone and progesterone with CYP106A1 and CYP21A2, respectively. Moreover, in view of the biotechnological application, the BmCPR is very promising, as it could be successfully utilized to establish CYP106A1- and CYP21A2-based whole-cell biotransformation systems, which yielded 0.3g/L hydroxy-testosterone products within 8h and 0.16g/L 21-hydroxyprogesterone within 6h, respectively. In conclusion, the BmCPR reported herein owns a great potential for further applications and studies and should be taken into consideration for bacterial and/or microsomal CYP-dependent bioconversions.

Plant membrane-protein mediated intracellular traffic of fatty acids and acyl lipids

In plants, de novo synthesis of fatty acids (FAs) occurs in plastids, whereas assembly and modification of acyl lipids is accomplished in the endoplasmic reticulum (ER) and plastids as well as in mitochondria. Subsequently, lipophilic compounds are distributed within the cell and delivered to their destination site. Thus, constant acyl-exchanges between subcellular compartments exist. These can occur via several modes of transport and plant membrane-intrinsic proteins for FA/lipid transfer have been shown to play an essential role in delivery and distribution. Lately, substantial progress has been made in identification and characterization of transport proteins for lipid compounds in plant organelle membranes. In this review, we focus on our current understanding of protein mediated lipid traffic between organelles of land plants.

Products of gut-microbial tryptophan metabolism inhibit the steroid hormone-synthesizing cytochrome P450 11A1.

Cytochromes P450 (P450s) are a superfamily of enzymes that catalyze the oxidative metabolism of a wide variety of xenobiotic chemical compounds, including drugs and carcinogens and play a key role in the synthesis of steroid hormones [1, 2]. The mitochondrial P450 11A1 (CYP11A1, EC 1.14.1.9), is a membrane-bound protein localized in the inner mitochondrial membrane of steroidogenic tissues such as the adrenal glands, gonads, and the gut [3], and commits the first and rate-limiting step of steroidogenesis, the side-chain cleavage of cholesterol to yield pregnenolone. First, cholesterol is hydroxlated at position C22 leading to the formation of 22(R)-hydroxycholesterol, which is then hydroxylated at position C20 to form 20(R), 22(R)-dihydroxy-cholesterol, followed by the oxidative cleavage of the bond between C20 and C22 resulting in pregnenolone [4]. The de novo production of steroid hormones such as the anti-inflammatory glucocorticoid cortisol in the gut is critical for maintenance of the intestinal homeostasis [5]. Disorders of intestinal steroidogenesis have been associated with inflammatory bowel diseases and are accompanied by lowered CYP11A1 expression [6], decreased glucocorticoid production [7], and cortisol-mediated expression of peroxisome proliferator-activated receptor gamma (PPARc) [8]. Indole and skatole are organic compounds formed as a product of tryptophan catabolism by gut bacteria, which serve as bacterial signaling molecules for a variety of biological actions [9]. In E. coli, indole is produced from tryptophan by the tryptophanase, which converts tryptophan into indole, pyruvate, and ammonia [10]. In human feces, indole concentrations have been reported to be between 0.25 and 1.1 mM [11]. The amount of indole in the gut greatly depends on exogenous tryptophan concentrations [12]. In this study, we describe an inhibitory effect of indole and skatole on CYP11A1, leading to a decrease in pregnenolone formation, the precursor of all mineralocorticoids, glucocorticoids, and sex steroids. Further, we demonstrate that both compounds serve as substrates for CYP11A1 and identify 5and 6-hydroxyindole as novel products. Additionally, using cell culture experiments, we provide evidence that exogenous indole is able to interact with CYP11A1 localized at the inner mitochondrial membrane.