Steroid Ring Research Articles

Synthesis of steroidal and nonsteroidal vicinal heterocyclic alcohols, N-(1-cycloalkenyl)heterocycles and their antibacterial studies

A solvent free steroidal and nonsteroidal epoxide ring opening reaction by nitrogen containing heterocycles under microwave irradiation is described. Some of the epoxide ring opening compounds were converted to their corresponding N-(1-cycloalkenyl)heterocycles via an acid catalyzed dehydration reaction. The antimicrobial activities of the epoxide ring opening compounds and N-(1-cycloalkenyl)heterocyclic compounds were tested by agar diffusion assay. Compounds 6, 9–12, 24 and 27 showed moderate inhibition against the growth of pathogenic bacteria Escherichia coli, Pseudomonas syringae, Bacillus subtilis, Proteus vulgaris and Staphylococcus aureus.

Mass Spectrometry Combinations for Structural Characterization of Sulfated-Steroid Metabolites

Steroid conjugates, which often occur as metabolites, are challenging to characterize. One application is female-mouse urine, where steroid conjugates serve as important ligands for the pheromone-sensing neurons. Although the two with the highest abundance in mouse urine were previously characterized with mass spectrometry (MS) and NMR to be sulfated steroids, many more exist but remain structurally unresolved. Given that their physical and chemical properties are similar, they are likely to have a sulfated steroid ring structure. Because these compounds occur in trace amounts in mouse urine and elsewhere, their characterization by NMR will be difficult. Thus, MS methods become the primary approach for determining structure. Here, we show that a combination of MS tools is effective for determining the structures of sulfated steroids. Using 4-pregnene analogs, we explored high-resolving power MS (HR-MS) to determine chemical formulae; HD exchange MS (HDX-MS) to determine number of active, exchangeable hydrogens (e.g., OH groups); methoxyamine hydrochloride (MOX) derivatization MS, or reactive desorption electrospray ionization with hydroxylamine to determine the number of carbonyl groups; and tandem MS (MS(n)), high-resolution tandem MS (HRMS/MS), and GC-MS to obtain structural details of the steroid ring. From the fragmentation studies, we deduced three major fragmentation rules for this class of sulfated steroids. We also show that a combined MS approach is effective for determining structure of steroid metabolites, with important implications for targeted metabolomics in general and for the study of mouse social communication in particular.

Functional analyses of three acyl‐CoA synthetases involved in bile acid degradation in Pseudomonas putida DOC21

Pseudomonas putida DOC21, a soil-dwelling proteobacterium, catabolizes a variety of steroids and bile acids. Transposon mutagenesis and bioinformatics analyses identified four clusters of steroid degradation (std) genes encoding a single catabolic pathway. The latter includes three predicted acyl-CoA synthetases encoded by stdA1, stdA2 and stdA3 respectively. The ΔstdA1 and ΔstdA2 deletion mutants were unable to assimilate cholate or other bile acids but grew well on testosterone or 4-androstene-3,17-dione (AD). In contrast, a ΔstdA3 mutant grew poorly in media containing either testosterone or AD. When cells were grown with succinate in the presence of cholate, ΔstdA1 accumulated Δ(1/4) -3-ketocholate and Δ(1,4) -3-ketocholate, whereas ΔstdA2 only accumulated 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC). When incubated with testosterone or bile acids, ΔstdA3 accumulated 3aα-H-4α(3'propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP) or the corresponding hydroxylated derivative. Biochemical analyses revealed that StdA1 converted cholate, 3-ketocholate, Δ(1/4) -3-ketocholate, and Δ(1,4) -3-ketocholate to their CoA thioesters, while StdA2 transformed DHOPDC to DHOPDC-CoA. In contrast, purified StdA3 catalysed the CoA thioesterification of HIP and its hydroxylated derivatives. Overall, StdA1, StdA2 and StdA3 are acyl-CoA synthetases required for the complete degradation of bile acids: StdA1 and StdA2 are involved in degrading the C-17 acyl chain, whereas StdA3 initiates degradation of the last two steroid rings. The study highlights differences in steroid catabolism between Proteobacteria and Actinobacteria.

Phytochemical Properties and Staining Ability of Red Onion (Allium cepa)Extract on Histological Sections

The staining ability of Allium cepa (red onion) skin extract on tissue sections was determined. The dried red onion skin was ground into small pieces and 300 g (150 g for aqueous and 150 g for ethanolic extraction) of the milled dried red onion skin was used for extraction. Dark red-pigmented granules were obtained. The aqueous and ethanolic extract of mordanted (Potassium aluminum alum and ferric chloride), non-mordanted and acidified solution prepared from the extract were used to stain tissue sections. Cytoplasm of cells and connective tissues were remarkably stained in shades of redish to yellowish brown colours. Preliminary phytochemical screening of these extracts reveals that it contained alkaloid steroid ring, saponin, Tannin, Flavonoid, Cardiac glycoside and anthraquinone. The presence of pigments, saponin, tannin, contributed to the staining ability of the extract.

Solid-state NMR study of fluorinated steroids

Solid-state {1H}13C cross-polarization/magic angle spinning (CP/MAS) NMR spectroscopy was performed to analyze two fluorinated steroids, i.e., betamethasone (BMS) and fludrocortisone acetate (FCA), that have fluorine attached to C9, as well as two non-fluorinated analogs, i.e., prednisolone (PRD) and hydrocortisone 21-acetate (HCA). The 13C signals of BMS revealed multiplet patterns with splittings of 16–215Hz, indicating multiple ring conformations, whereas the 13C signals of FCA, HCA, and PRD exhibited only singlet patterns, implying a unique conformation. In addition, BMS and FCA exhibited substantial deviation (>3.5ppm) in approximately half of the 13C signals and significant deviation (>45ppm) in the 13C9 signal compared to PRD and HCA, respectively. In this study, we demonstrate that fluorinated steroids, such as BMS and FCA, have steroidal ring conformation(s) that are distinct from non-fluorinated analogs, such as PRD and HCA.

Preliminary phytochemical screening of methanolic–aqua extract of Acanthospermum australe leaves.

The study was carried to analyze the phytochemical constituents of the Acanthospermum australe. The plant samples were extracted using methanol and water in the ratio of 9:1. From the study the extract of Acanthospermum australe was found to contain tannins, saponins, terpenoids, phenols, alkaloids, steroidal rings but steroids and flavonoids were found to be absent. The presence of these important phytochemicals in the plant roots is a scientific justification of the plant use in the traditional treatment against various diseases affecting humans and animals. However, more research needs to be done to identify the specific compounds, their structural formulas and contribution towards medicinal values of the plant.

Phytopharmacological analysis of methanolic-aqua extract (fractions) of senna didymobotrya roots.

The study was carried to analyze the phytochemical constituents of the Senna didymobotrya roots. The plant samples were extracted using methanol and water in the ratio of 9:1. The crude extract was disintegrated using different solvents according to their polarity. The solvents used were chloroform, ethyl acetate, butanol and water respectively. All the extracts were subjected to phytochemical study using standard procedures. From the study the of crude extract of Senna didymobotrya was found to contain tannins, saponins, terpenoids, flavonoids, phenols, alkaloids, steroidal rings but steroids were found to be absent. The presence of these important phytochemicals in the plant roots is a scientific justification of the use of the plant in the traditional treatment against various diseases affecting humans and animals. However, more research needs to be done to identify the specific compounds, there structural formulas and their medicinal values.

The Anti‐Bredt Red Flag! Reassignment of Neoveratrenone

AbstractThe bicyclo[3.3.1]non‐1‐en‐3‐one motif, which has yet to be observed in natural products, was recently reported within the skeleton of neoveratrenone. On the basis of Bredt's rule, a plausible reassignment matching closely the C‐nor‐D‐homo steroidal ring system has been proposed herein.

Inhibitory effect of oxygenated cholestan-3β-ol derivatives on the growth of Mycobacterium tuberculosis

A variety of cholestan-3β-ol derivatives, which are oxygenated at different positions of the steroid ring system, were prepared and tested for their inhibition of the Mycobacterium tuberculosis H37Rv strain. Several compounds showed significant antitubercular activities with MIC90 values in the range 4–8μM and low or non-detectable toxicity against mammalian cells.

Lyotropic Liquid Crystalline Phases of a Phytosterol Ethoxylate in Amide Solvents

Materials exhibiting unique aggregation behavior in nonaqueous solvents have attracted attention due to their wide applications. Motivated by this recent interest, the aggregation properties of a phytosterol ethoxylate surfactant, BPS-10, in three organic amide compounds, formamide (FA), N-methylformamide (NMF), and N,N-dimethyl- formamide (DMF), have been studied. Polarized optical microscopy and small-angle X-ray scattering techniques were used to investigate the lyotropic liquid crystalline (LLC) phases formed in these binary systems. Herein, we discuss the relationship between subtle intermolecular interactions and the aggregation behavior of BPS-10. As good proton donors or acceptors to form hydrogen bonding, FA molecules allow BPS-10 to show a richer phase behavior. Compared with the systems formed in water and ionic liquids, the LLCs constructed in FA have higher thermal stability. In addition, two kinds of lamellar phases could coexist in a narrow region. With the methyl replacement in formamide, however, the ability to form hydrogen bonds is reduced and the solvent bulk phase structure becomes less ordered from FA to DMF. Consequently, the solvophobic interaction of BPS-10 becomes weaker, and the LLCs are more difficult to form. In addition, the extra strong interactions between the steroid rings of BPS-10 may provide enough driving force to produce the hexagonal phase (H1) directly in NMF and DMF without micelle formation, thereby creating a novel sequence (isotropic → H1 → Lα) of ordered phases with increasing surfactant concentration. The results discussed herein should prove to be a useful complement to the growing body of literature regarding steroid surfactant aggregation in polar organic solvents.

Carcinogenic liver fluke Opisthorchis viverrini oxysterols detected by LC–MS/MS survey of soluble fraction parasite extract

Liquid chromatography in tandem mass spectrometry (LC–MS/MS) has emerged as an informative tool to investigate oxysterols (oxidized derivatives of cholesterol) in helminth parasite associated cancers. Here, we used LC–MS/MS to investigate in soluble extracts of the adult developmental stage of Opisthorchis viverrini from experimentally infected hamsters. Using comparisons with known bile acids and the metabolites of estrogens, the LC–MS data indicated the existence of novel oxysterol derivatives in O. viverrini. Most of these derivatives were ramified at C-17, in similar fashion to bile acids and their conjugated salts. Several were compatible with the presence of an estrogen core, and/or hydroxylation of the steroid aromatic ring A, hydroxylation of both C-2 and C-3 of the steroid ring and further oxidation into an estradiol-2,3-quinone.

Thermodynamic and solution state NMR characterization of the binding of secondary and conjugated bile acids to STARD5

STARD5 is a member of the STARD4 sub-family of START domain containing proteins specialized in the non-vesicular transport of lipids and sterols. We recently reported that STARD5 binds primary bile acids. Herein, we report on the biophysical and structural characterization of the binding of secondary and conjugated bile acids by STARD5 at physiological concentrations. We found that the absence of the 7α-OH group and its epimerization increase the affinity of secondary bile acids for STARD5. According to NMR titration and molecular modeling, the affinity depends mainly on the number and positions of the steroid ring hydroxyl groups and to a lesser extent on the presence or type of bile acid side-chain conjugation. Primary and secondary bile acids have different binding modes and display different positioning within the STARD5 binding pocket. The relative STARD5 affinity for the different bile acids studied is: DCA>LCA>CDCA>GDCA>TDCA>CA>UDCA. TCA and GCA do not bind significantly to STARD5. The impact of the ligand chemical structure on the thermodynamics of binding is discussed. The discovery of these new ligands suggests that STARD5 is involved in the cellular response elicited by bile acids and offers many entry points to decipher its physiological role.

Direct Photodegradation of Androstenedione and Testosterone in Natural Sunlight: Inhibition by Dissolved Organic Matter and Reduction of Endocrine Disrupting Potential

In surface waters, two of the most commonly observed androgenic steroid hormones are androstenedione (AD) and testosterone (T). This study compares the photodegradation of dilute (<10 μg L(-1)) aqueous solutions of AD and T in natural sunlight, and evaluates the endocrine-disrupting potential of the resulting solutions. This study also examines the effect of dissolved organic matter (DOM) on AD photodegradation. During spring and summer at Henderson, NV, USA (latitude 36.04°N), AD and T underwent direct photodegradation, with half-lives ranging from 3.7 to 10.8 h. In three model DOM solutions, AD's half-life increased by 11% to 35%. Using screening factors to eliminate DOM's inner filter effect, quantum yield calculations suggested that light screening was primarily responsible for AD's increased half-life, and that physical quenching further inhibited AD's photodegradation in two out of three DOM solutions. In vitro androgenic activity of the AD and T solutions decreased approximately as fast as AD and T were removed, suggesting that solar photodegradation reduces the risk of endocrine disruption in surface waters impacted by AD or T, subject to continuing inputs. Reduced in vitro androgenic activity appears to be related to steroid ring cleavage and the formation of highly oxidized photoproducts.

Phytochemical and bioactivity evaluation of Senna didymobotrya fresen irwin used by the Nandi community in Kenya

Medicinal plants play a vital role in the traditional treatment of various diseases in Africa and the world at large. In Nandi county rift valley province in Kenya Senna didymobotyya is one of the most valued plants traditionally, this is mainly attributed to the famous mursik milk in which the plant is used as a preservative. A study was carried out to investigate the bioactivity of the plant leaves. Organism tested were Escherichia coli, Salmonella typhi, Enterobacter aerogenes , Klebsiella sp., Streptococcus pyogenes isolated from clinical specimens, and Proteus vulgaris and Bacillus cereus , obtained as standard isolates from commercial source. From the study Senna didymobotrya was found to contain secondary metabolites, saponins, flavonoids, tannins, phenols, steroids, steroidal ring (glycone portions of the glycoside), steroidal nucleus (glycone portion of the glycoside) and cardiac glycosides. The plant leaves were found not to contain alkaloids, terpenoids and anthroquinones. The bioactivity of the plant extract observed in this study can be attributed to the presence of these phytochemicals. Results from the bioassay Senna extract showed the highest zone of inhibition was obtained against Bacillus cereus at 500 mg/ml and its minimum inhibitory concentration was 31.25 mg/ml. The extract was also active against all the organisms at 500 mg/ml. Its minimum inhibitory concentration was 250 mg/ml for Streptococcus pyogenes , Escherichia coli and Proteus vulgaris .

The Essential Function of Genes for a Hydratase and an Aldehyde Dehydrogenase for Growth of Pseudomonas sp. Strain Chol1 with the Steroid Compound Cholate Indicates an Aldolytic Reaction Step for Deacetylation of the Side Chain

In the bacterial degradation of steroid compounds, the enzymes initiating the breakdown of the steroid rings are well known, while the reactions for degrading steroid side chains attached to C-17 are largely unknown. A recent in vitro analysis with Pseudomonas sp. strain Chol1 has shown that the degradation of the C5 acyl side chain of the C24 steroid compound cholate involves the C22 intermediate 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20S-carbaldehyde (DHOPDCA) with a terminal aldehyde group. In the present study, candidate genes with plausible functions in the formation and degradation of this aldehyde were identified. All deletion mutants were defective in growth with cholate but could transform it into dead-end metabolites. A mutant with a deletion of the shy gene, encoding a putative enoyl coenzyme A (CoA) hydratase, accumulated the C24 steroid (22E)-7α,12α-dihydroxy-3-oxochola-1,4,22-triene-24-oate (DHOCTO). Deletion of the sal gene, formerly annotated as the steroid ketothiolase gene skt, resulted in the accumulation of 7α,12α,22-trihydroxy-3-oxochola-1,4-diene-24-oate (THOCDO). In cell extracts of strain Chol1, THOCDO was converted into DHOPDCA in a coenzyme A- and ATP-dependent reaction. A sad deletion mutant accumulated DHOPDCA, and expression in Escherichia coli revealed that sad encodes an aldehyde dehydrogenase for oxidizing DHOPDCA to the corresponding acid 7α,12α-dihydroxy-3-oxopregna-1,4-diene-20-carboxylate (DHOPDC) with NAD(+) as the electron acceptor. These results clearly show that the degradation of the acyl side chain of cholate proceeds via an aldolytic cleavage of an acetyl residue; they exclude a thiolytic cleavage for this reaction step. Based on these results and on sequence alignments with predicted aldolases from other bacteria, we conclude that the enzyme encoded by sal catalyzes this aldolytic cleavage.

Anaerobic and aerobic cleavage of the steroid core ring structure by Steroidobacter denitrificans

The aerobic degradation of steroids by bacteria has been studied in some detail. In contrast, only little is known about the anaerobic steroid catabolism. Steroidobacter denitrificans can utilize testosterone under both oxic and anoxic conditions. By conducting metabolomic investigations, we demonstrated that S. denitrificans adopts the 9,10-seco-pathway to degrade testosterone under oxic conditions. This pathway depends on the use of oxygenases for oxygenolytic ring fission. Conversely, the detected degradation intermediates under anoxic conditions suggest a novel, oxygenase-independent testosterone catabolic pathway, the 2,3-seco-pathway, which differs significantly from the aerobic route. In this anaerobic pathway, testosterone is first transformed to 1-dehydrotestosterone, which is then reduced to produce 1-testosterone followed by water addition to the C-1/C-2 double bond of 1-testosterone. Subsequently, the C-1 hydroxyl group is oxidized to produce 17-hydroxy-androstan-1,3-dione. The A-ring of this compound is cleaved by hydrolysis as evidenced by H2(18)O-incorporation experiments. Regardless of the growth conditions, testosterone is initially transformed to 1-dehydrotestosterone. This intermediate is a divergence point at which the downstream degradation pathway is governed by oxygen availability. Our results shed light into the previously unknown cleavage of the sterane ring structure without oxygen. We show that, under anoxic conditions, the microbial cleavage of steroidal core ring system begins at the A-ring.

Mycobacterium tuberculosis Utilizes a Unique Heterotetrameric Structure for Dehydrogenation of the Cholesterol Side Chain

Compounding evidence supports the important role in pathogenesis that the metabolism of cholesterol by Mycobacterium tuberculosis plays. Elucidating the pathway by which cholesterol is catabolized is necessary to understand the molecular mechanism by which this pathway contributes to infection. On the basis of early metabolite identification studies in multiple actinomycetes, it has been proposed that cholesterol side chain metabolism requires one or more acyl-CoA dehydrogenases (ACADs). There are 35 genes annotated as encoding ACADs in the M. tuberculosis genome. Here we characterize a heteromeric ACAD encoded by Rv3544c and Rv3543c, formerly named fadE28 and fadE29, respectively. We now refer to genes Rv3544c and Rv3543c as chsE1 and chsE2, respectively, in recognition of their validated activity in cholesterol side chain dehydrogenation. Analytical ultracentrifugation and liquid chromatography-ultraviolet experiments establish that ChsE1-ChsE2 forms an α(2)β(2) heterotetramer, a new architecture for an ACAD. Our bioinformatic analysis and mutagenesis studies reveal that heterotetrameric ChsE1-ChsE2 has only two active sites. E241 in ChsE2 is required for catalysis of dehydrogenation by ChsE1-ChsE2. Steady state kinetic analysis establishes the enzyme is specific for an intact steroid ring system versus hexahydroindanone substrates with specificity constants (k(cat)/K(M)) of (2.5 ± 0.5) × 10(5) s(-1) M(-1) versus 9.8 × 10(2) s(-1) M(-1), respectively, at pH 8.5. The characterization of a unique ACAD quaternary structure involved in sterol metabolism that is encoded by two distinct cistronic ACAD genes opens the way to identification of additional sterol-metabolizing ACADs in M. tuberculosis and other actinomycetes through bioinformatic analysis.

Relationship of serum estrogens and estrogen metabolites to postmenopausal breast cancer risk: a nested case-control study

IntroductionElevated levels of circulating estrogens are linked to breast cancer risk among postmenopausal women but little is known about the importance of estrogen metabolism. A recently developed liquid chromatography tandem mass spectrometry-based method (LC-MS/MS) measuring a panel of 15 estrogen metabolites (EM) has been evaluated in one study, linking high levels of 2-pathway metabolites relative to the parent estrogens to reduced breast cancer risk. We analyzed this panel of EM in a nested case-control study of postmenopausal breast cancer.MethodsBetween 1977 and 1987, 6,915 women provided blood samples to the Columbia Missouri Serum Bank and were followed for incident breast cancer through December 2002. We studied 215 postmenopausal breast cancer cases and 215 matched controls who were postmenopausal and not using exogenous hormones at the time of blood draw. EM were examined individually, grouped by pathway (hydroxylation at the C-2, C-4 or C-16 positions of the steroid ring) and by ratios of the groupings. Logistic regression models controlling for matching and breast cancer risk factors were used to calculate quartile-specific odds ratios (ORs) and 95% CIs.ResultsSignificant elevated risks were not observed for individual EM, except for quartiles of 16-epiestriol (P trend = 0.07). The OR for total EM, the parent estrogens estrone and estradiol, and 2-pathway catechol EM (2-hydroxyestrone and 2-hydroxyestradiol) were elevated but the trends were not statistically significant. Among 2-pathway metabolites, risks for the highest levels of 2-hydroxyestrone-3-methyl ether and 2-methoxyestradiol were reduced; ORs for women in the highest versus lowest quartiles were 0.57 (95% CI = 0.33 to 0.99) and 0.53 (95% CI = 0.30 to 0.96), respectively. Overall, women with higher levels of 2-pathway EM had a reduced risk of breast cancer, which remained after accounting for levels of parent EM, 4-pathway EM and 16-pathway EM (all trends, P <0.11).ConclusionsWomen with more extensive hydroxylation along the 2-pathway may have a reduced risk of postmenopausal breast cancer. Further studies are needed to clarify the risks for specific EM and complex patterns of estrogen metabolism. This will require aggregation of EM results from several studies.

Properties of Membranes Derived from the Total Lipids Extracted from the Human Lens Cortex and Nucleus

The organization and physical properties of lens lipid membranes made from the total lipids extracted from the human lens cortex and nucleus of 41- to 60-year-old donors were investigated using electron paramagnetic resonance (EPR) spin-labeling. Profiles of the phospholipid alkyl-chain order, fluidity, oxygen transport parameter, and hydrophobicity were assessed across membranes and coexisting membrane domains. Lens lipid membranes prepared from the lens cortex and nucleus were found to contain two distinct lipid environments, which were assigned as the bulk phospholipid-cholesterol domain and the cholesterol bilayer domain (CBD). The alkyl chains of phospholipids were strongly ordered at all depths, indicating that the amplitude of the wobbling motion of alkyl chains was small. However, profiles of the membrane fluidity, which explicitly contain time (expressed as the spin-lattice relaxation rate) and depend on the rotational motion of spin labels, show relatively high fluidity of alkyl chains close to the membrane center. Profiles of the oxygen transport parameter and hydrophobicity have a rectangular shape with an abrupt change between the C9 and C10 positions, which is approximately where the steroid ring structure of cholesterol reaches into the membrane. The amount of CBD was greater in nuclear membranes than in cortical membranes. The presence of the CBD in lens lipid membranes, which at 37°C showed a permeability coefficient for oxygen about 60% smaller than across a water layer of the same thickness, would be expected to raise the barrier for oxygen transport across the fiber cell membrane.

FadD3 is an acyl‐CoA synthetase that initiates catabolism of cholesterol rings C and D in actinobacteria

The cholesterol catabolic pathway occurs in most mycolic acid-containing actinobacteria, such as Rhodococcus jostii RHA1, and is critical for Mycobacterium tuberculosis (Mtb) during infection. FadD3 is one of four predicted acyl-CoA synthetases potentially involved in cholesterol catabolism. A ΔfadD3 mutant of RHA1 grew on cholesterol to half the yield of wild-type and accumulated 3aα-H-4α(3'-propanoate)-7aβ-methylhexahydro-1,5-indanedione (HIP), consistent with the catabolism of half the steroid molecule. This phenotype was rescued by fadD3 of Mtb. Moreover, RHA1 but not ΔfadD3 grew on HIP. Purified FadD3(Mtb) catalysed the ATP-dependent CoA thioesterification of HIP and its hydroxylated analogues, 5α-OH HIP and 1β-OH HIP. The apparent specificity constant (k(cat) /K(m) ) of FadD3(Mtb) for HIP was 7.3 ± 0.3 × 10(5) M(-1) s(-1) , 165 times higher than for 5α-OH HIP, while the apparent K(m) for CoASH was 110 ± 10 μM. In contrast to enzymes involved in the catabolism of rings A and B, FadD3(Mtb) did not detectably transform a metabolite with a partially degraded C17 side-chain. Overall, these results indicate that FadD3 is a HIP-CoA synthetase that initiates catabolism of steroid rings C and D after side-chain degradation is complete. These findings are consistent with the actinobacterial kstR2 regulon encoding ring C/D degradation enzymes.