Degradation of Bile Acids by Soil and Water Bacteria

Bile acids are surface-active steroid compounds with a C5 carboxylic side chain at the steroid nucleus. They are produced by vertebrates, mainly functioning as emulsifiers for lipophilic nutrients, as signaling compounds, and as an antimicrobial barrier in the duodenum. Upon excretion into soil and water, bile acids serve as carbon- and energy-rich growth substrates for diverse heterotrophic bacteria. Metabolic pathways for the degradation of bile acids are predominantly studied in individual strains of the genera Pseudomonas, Comamonas, Sphingobium, Azoarcus, and Rhodococcus. Bile acid degradation is initiated by oxidative reactions of the steroid skeleton at ring A and degradation of the carboxylic side chain before the steroid nucleus is broken down into central metabolic intermediates for biomass and energy production. This review summarizes the current biochemical and genetic knowledge on aerobic and anaerobic degradation of bile acids by soil and water bacteria. In addition, ecological and applied aspects are addressed, including resistance mechanisms against the toxic effects of bile acids.

Highly Hydrophilic and Lipophilic Derivatives of Bile Salts

Lipophilicity of 15 derivatives of sodium cholate, defined by the octan-1-ol/water partition coefficient (log P), has been theoretically determined by the Virtual log P method. These derivatives bear highly hydrophobic or highly hydrophilic substituents at the C3 position of the steroid nucleus, being linked to it through an amide bond. The difference between the maximum value of log P and the minimum one is enlarged to 3.5. The partition coefficient and the critical micelle concentration (cmc) are tightly related by a double-logarithm relationship (VirtuallogP=−(1.00±0.09)log(cmcmM)+(2.79±0.09)), meaning that the Gibbs free energies for the transfer of a bile anion from water to either a micelle or to octan-1-ol differ by a constant. The equation also means that cmc can be used as a measurement of lipophilicity. The demicellization of the aggregates formed by three derivatives of sodium cholate bearing bulky hydrophobic substituents has been studied by surface tension and isothermal titration calorimetry. Aggregation numbers, enthalpies, free energies, entropies, and heat capacities, ΔCP,demic, were obtained. ΔCP,demic, being positive, means that the interior of the aggregates is hydrophobic.

Estrogens and antiestrogens. Modern synthetic approaches to directed modification of estra-1,3,5(10)-triene steroids: goals, reactions, and methods

The monograph summarizes the information over the past 20 years on the currently widely used and promising methods for the synthesis of estra-1,3,5(10)-triene derivatives by modifying natural estrogens - estrone and estradiol. The main practical goals of modifying this class of steroids and achievements in the chemistry of steroidal antiestrogens, which are promising drugs for hormonal therapy, are considered. Special attention is paid to the stereochemical features of the reactions and the specific problems of modification of the steroid nucleus of estratrienes associated with the presence of an aromatic fragment in their structure. In addition, the data on the reactivity and stereochemical aspects of the transformations of 13-epiestratriene steroids were summarized. The monograph is intended for a wide range of specialists in the field of organic synthesis, organic, bioorganic, and medicinal chemistry.

Further Studies on the 3-Ketosteroid 9α-Hydroxylase of Rhodococcus ruber Chol-4, a Rieske Oxygenase of the Steroid Degradation Pathway

The biochemistry and genetics of the bacterial steroid catabolism have been extensively studied during the last years and their findings have been essential to the development of biotechnological applications. For instance, metabolic engineering of the steroid-eater strains has allowed to obtain intermediaries of industrial value. However, there are still some drawbacks that must be overcome, such as the redundancy of the steroid catabolism genes in the genome and a better knowledge of its genetic regulation. KshABs and KstDs are key enzymes involved in the aerobic breakage of the steroid nucleus. Rhodococcus ruber Chol-4 contains three kshAs genes, a single kshB gene and three kstDs genes within its genome. In the present work, the growth of R. ruber ΔkshA strains was evaluated on different steroids substrates; the promoter regions of these genes were analyzed; and their expression was followed by qRT-PCR in both wild type and ksh mutants. Additionally, the transcription level of the kstDs genes was studied in the ksh mutants. The results show that KshA2B and KshA1B are involved in AD metabolism, while KshA3B and KshA1B contribute to the cholesterol metabolism in R. ruber. In the kshA single mutants, expression of the remaining kshA and kstD genes is re-organized to survive on the steroid substrate. These data give insight into the fine regulation of steroid genes when several isoforms are present.

Construction and Functional Analysis of Mycolicibacterium smegmatis Recombinant Strains Carrying the Genes of Bacillary Cytochromes CYP106A1 and CYP106A2

Mycolicibacterium smegmatis mc2155 has been genetically modified to be used as a platform for the expression of foreign cytochrome P450 monooxygenases by introducing deletions in the kshB and kstD genes that encode key stages of the enzymatic destruction of the steroid nucleus. Three sets of genetic constructs have been created for heterologous expression of the genes of cytochromes P450 CYP106A1 from Bacillus megaterium DSM319 and CYP106A2 from Bacillus megaterium ATCC13368 in Mycolicibacterium smegmatis mc2155 (ΔkshBΔkstD) cells. The recombinant plasmids contained monocistronic expression cassettes of cytochrome genes (NS31 and pNS32), or tricistronic cassettes of cytochrome genes together with cDNA copies of adrenodoxin and andrenodoxin reductase genes of the bovine adrenal cortex (pNS33 and pNS34), or monocistronic gene cassettes of chimeric cytochromes fused with the DNA sequence encoding the CYP116B2 reductase domain from the soil bacterium Rhodococcus sp. NCIMB 9784 (pNS35 and pNS36). The recombinant strains of mycolicibacteria were shown to selectively monohydroxylate androstenedione (AD) under growth conditions. The product was identified as 15-hydroxyandrostenedione (15-OH-AD) by mass spectrometry and 1H and 13C NMR spectroscopy. The maximum level of 15-OH-AD production (17.3 ± 1.5 mg/L) was observed when using the recombinant M. smegmatis mc2155 (ΔkshBΔkstD) (pNS32) strain, which expresses a single cyp106A2 gene from B. megaterium ATCC13368. Host proteins of M. smegmatis mc2155 were shown to be capable of supplying electrons to heterologous cytochromes to support their hydroxylating activity. The results are of priority character, expand the understanding of the hydroxylation of steroid compounds by bacterial cytochromes CYP106A1/A2 and are important for the creation of microbial strains producing valuable hydroxysteroids. cyp106A1, cyp106A2, cytochrome P450, heterologous expression, Bacillus megaterium, Mycolicibacterium smegmatis, 15β-hydroxylation, bioconversion, steroids This work was supported by the Russian Science Foundation (project No. 21-64-00024).

Synthesis, NMR Characterization, and Antileukemic Activity of N-Nonanoylpiperazinyl-5α-Androstane-3α,17β-Diol A-Ring Derivatives

The combination of an androstane-3,17-diol nucleus and a 2β-N-alkylamidopiperazino sidechain is important for the anticancer activity of a new family of steroid derivatives. As the structure-activity relationship studies have so far been limited to the beta orientation of the substituent at position 2 of the steroid nucleus, a series of analogs (compounds 1–4) were synthesized to investigate the impact on biological activity of A-ring substitution. Nuclear magnetic resonance (NMR) analysis, especially using a series of 2D experiments, such as correlation spectroscopy (COSY), homonuclear Overhauser effect spectroscopy (NOESY), heteronuclear single-quantum correlation (HSQC), and heteronuclear multiple-bond correlation (HMBC) provided crucial information that was found essential in confirming the sidechain position and orientation of compounds 1–4. Assessment of their antiproliferative activity on leukemia HL-60 cells confirmed the best efficiency of the 2β-sidechain/3α-OH orientation (compound 1) compared to the other configurations tested (compounds 2–4).

Diosgenin-3,6-dione: second polymorph in space group P212121

The title steroid, [(25R)-spirost-4-en-3,6-dione, C27H38O4], is obtained by oxidation of diosgenin, using the Jones reagent (CrO3/H2SO4). The crystal structure was previously reported in space group P212121, but nonetheless with the wrong absolute configuration and omitting positions for H atoms [Rajnikant et al. (2000). Mol. Cryst. Liq. Cryst. Sci. Technol. Sect. C, 12, 101–110]. The diffraction data set reported herein is for a second polymorph in the same space group, as evidenced by simulated powder patterns. Both forms are characterized by a similar orthorhombic unit cell, and a similar arrangement of the molecules in the crystal structure. However, the conformation of the A/B rings in the steroid nucleus is slightly modified, leading to the observed polymorphism.

Δ1-Dehydrogenation and C20 Reduction of Cortisone and Hydrocortisone Catalyzed by Rhodococcus Strains

Prednisone and prednisolone are steroids widely used as anti-inflammatory drugs. Development of the pharmaceutical industry is currently aimed at introducing biotechnological processes and replacing multiple-stage chemical syntheses. In this work we evaluated the ability of bacteria belonging to the Rhodococcus genus to biotransform substrates, such as cortisone and hydrocortisone, to obtain prednisone and prednisolone, respectively. These products are of great interest from a pharmaceutical point of view as they have higher anti-inflammatory activity than the starting substrates. After an initial lab-scale screening of 13 Rhodococcus strains, to select the highest producers of prednisone and prednisolone, we reported the 200 ml-batch scale-up to test the process efficiency and productivity of the most promising Rhodococcus strains. R. ruber, R. globerulus and R. coprophilus gave the Δ1-dehydrogenation products of cortisone and hydrocortisone (prednisone and prednisolone) in variable amounts. In these biotransformations, the formation of products with the reduced carbonyl group in position C20 of the lateral chain of the steroid nucleus was also observed (i.e., 20β-hydroxy-prednisone and 20β-hydroxy-prednisolone). The yields, the absence of collateral products, and in some cases the absence of starting products allow us to say that cortisone and hydrocortisone are partly degraded.

Design, Synthesis, and Biological Evaluation of Two Series of Novel A-Ring Fused Steroidal Pyrazines as Potential Anticancer Agents

Background: Increasingly, different heterocyclic systems have been introduced into the steroid nucleus to significantly enhance the antitumor activities of steroid molecules. However, in this study, few literature precedents describing the pyrazine heterocyclic-condensed modification to an A-ring of steroid monomers were found, although the pyrazine group is thought to be essential for the potent anticancer activity of clinically relevant drugs and natural steroid dimers. Methods and Results: Two series of novel A-ring fused steroidal pyrazines were designed and efficiently synthesized from commercially available progesterone via key α-ketoenol intermediates. Through a cell counting kit-8 cytotoxic assay of 36 derivatives for three tumor cells, 14 compounds displayed significant antiproliferative activity compared to 5-fluorouracil, especially for human prostatic tumor cells (PC-3) in vitro. Further mechanistic studies indicated that the most active compound, 12n (IC50, 0.93 μM; SI, 28.71), could induce the cell apoptosis of PC-3 cells in a dose-dependent manner and cause cell cycle arrest in the G2/M phase. The molecular docking study suggested that compound 12n fitted the active sites of cytochrome P450 17A1 (6CIZ) well. Conclusions: 12n might serve as a promising lead compound for the development of novel anticancer drugs. This facile ring-closing strategy may provide a novel and promising avenue for the cycloaddition reaction of the steroidal skeleton through α-ketoenol intermediates.

Distinct Regioselectivity of Fungal P450 Enzymes for Steroidal Hydroxylation

ABSTRACT In this study, we identified two P450 enzymes (CYP5150AP3 and CYP5150AN1) from Thanatephorus cucumeris NBRC 6298 by combination of transcriptome sequencing and heterologous expression in Pichia pastoris. The biotransformation of 11-deoxycortisol and testosterone by Pichia pastoris whole cells coexpressing the cyp5150ap3 and por genes demonstrated that the CYP5150AP3 enzyme possessed steroidal 7β-hydroxylase activities toward these substrates, and the regioselectivity was dependent on the structures of steroidal compounds. CYP5150AN1 catalyzed the 2β-hydroxylation of 11-deoxycortisol. It is interesting that they display different regioselectivity of hydroxylation from that of their isoenzyme, CYP5150AP2, which possesses 19- and 11β-hydroxylase activities. IMPORTANCE The steroidal hydroxylases CYP5150AP3 and CYP5150AN1 together with the previously characterized CYP5150AP2 belong to the CYP5150A family of P450 enzymes with high amino acid sequence identity, but they showed completely different regioselectivities toward 11-deoxycortisol, suggesting the regioselectivity diversity of steroidal hydroxylases of CYP5150 family. They are also distinct from the known bacterial and fungal steroidal hydroxylases in substrate specificity and regioselectivity. Biocatalytic hydroxylation is one of the important transformations for the functionalization of steroid nucleus rings but remains a very challenging task in organic synthesis. These hydroxylases are useful additions to the toolbox of hydroxylase enzymes for the functionalization of steroids at various positions.