Hydroxylation Of Progesterone Research Articles

A Novel A105Y Mutant of CYP17A1 Exhibits Almost Perfect Regioselectivity in the Production of 17α-Hydroxyprogesterone.

17α-Hydroxyprogesterone (17α-OHP) is a steroid hormone with significant biological activity that can be obtained by catalyzing progesterone (PROG), the main product of sitosterol, through CYP17A1. However, increasing the catalytic specificity of HCYP17A1 for C17 hydroxylation of progesterone (PROG) poses a formidable challenge due to the close proximity of the C16 and C17 positions. In this study, a rational design was utilized to alter the spatial configuration of the substrate channel, leading to the complete abolition of its 16-hydroxylation activity. Subsequent molecular dynamics simulations revealed that the A105Y mutation heightened the rigidity of the G95-I112 region of CYP17A1, consequently regulating the direction of the entry of PROG into the catalytic pocket. Moreover, the establishment of hydrogen bonding between Y105 and R239, coupled with Pi-stacking of A105Y with F114, effectively immobilizes the substrate PROG in a fixed position, explaining the practically perfect regioselectivity observed in A105Y. Finally, a multifaceted enzymatic cascade system, incorporating A105Y, cytochrome P450 reductase (CPR), and glucose-6-phosphate dehydrogenase (ZWF) for NADPH cofactor regeneration, was constructed in Pichia pastoris GS115. The resulting biocatalyst produced 106 ± 3.2 mg L-1 17α-OHP, a 4.6-fold increase compared with A105Y alone. Thus, this study provides valuable insights for improving the regioselectivity and activity of P450 enzymes.

Biocatalytic Hydroxylation of Progesterone by Penicillium aculeatum

Background: Biotransformation is a powerful process for producing steroid compounds, and fungi are commonly regarded as effective biological agents for this purpose. They facilitate reactions that are difficult to perform via conventional chemical methods. Objective: In the current study, the ability of Penicillium aculeatum to biotransform progesterone was studied. Methods: Forty-eight hours after the incubation of active P. aculeatum with substrate (progesterone), the reaction medium was extracted and chromatography methods isolated metabolites. The chemical structures of the products were characterized by various spectroscopic techniques. Results: Two main hydroxylated products, 14α-hydroxyprogesterone, and 7α,14α-dihydroxyprogesterone, were finally identified. Conclusion: P. aculeatum may be considered a functional biocatalyst for some biotransformation processes.

Optical annealing of peroxo-ferric intermediates in CYP17A1 and product formation.

Human cytochrome P450 CYP17A1 catalyzes the hydroxylation of pregnenolone and progesterone at the C17 position, with subsequent C17-C20 bond scission, to form dehydroepiandrosterone and androstenedione respectively. The first hydroxylation reaction is faster in H2O than in D2O, while the second carbon‑carbon bond scission event demonstrates an inverse solvent isotope effect, which is more pronounced for 17-hydroxy pregnenolone. In order to better understand the cause of this difference, we compared the optical absorption spectra of oxygenated CYP17A1 with the four substrates (pregnenolone, progesterone, 17-hydroxy pregnenolone and 17-hydroxy progesterone) in both H2O and D2O. We also studied the temperature-dependent decay of the peroxo-ferric and hydroperoxo-ferric intermediates generated by cryoradiolysis of the corresponding oxygenated heme proteins at 77 K. For both pregnenolone and 17-hydroxypregnenolone, annealing of the peroxo-intermediates was observed at lower temperatures in H2O than in D2O. In contrast, no solvent isotope effect was detected when progesterone or 17-hydroxyprogesterone were used as substrates. These differences are attributed to their different positioning in the P450 active site with respect to the heme bound peroxo (Fe-OO−) moiety, which is in agreement with earlier structural and spectroscopic investigations. Analysis of the samples run in both H2O and in D2O, where 17-hydroxyprogesterone is the substrate, demonstrated significant (∼25%) yield of androstenedione product relative to the oxygenated starting material.

Production of 14α-Hydroxy Progesterone Using a Steroidal Hydroxylase from Cochliobolus lunatus Expressed in Escherichia coli

Steroids with hydroxylation at C14 are drawing increased attention because of their diverse biological activities and applications. P-450lun from Cochliobolus lunatus is the first fungal cytochrome P450 reported to have 14α-hydroxylase activity. Studies have shown that P-450lun catalyzes the hydroxylation of progesterone (PROG) at C14α with low regiospecificity and activity. To improve its regiospecificity and activity for PROG, truncated forms of P-450lun and its cognate redox partner CPRlun were functionally co-expressed in Escherichia coli. Then, a semi-rational protein engineering approach was applied to P-450lun, resulting in a double-site mutant E109A/F297W with enhanced 14α-position selectivity for PROG compared with the wild-type P-450lun (97% vs. 28%). Protein structure analysis revealed that the F297W substitution can hinder the binding pose for 11β-hydroxylation product formation. Finally, whole-cell catalysis was optimized, and the final titer of 14α-OH-PROG reached 16.0 mg/L. This is the first report where a fungal 14α-hydroxylase was functionally expressed in Escherichia coli. The steroid hydroxylation system obtained in this study can serve as a basis for the synthesis of 14α-hydroxylated PROG and the rapid evolution of eukaryotic cytochrome P-450lun.

Tracking protein-protein interactions by NMR: conformational selection in human steroidogenic cytochrome P450 CYP17A1 induced by cytochrome b5.

The human steroidogenic cytochrome P450 CYP17A1 catalyzes two types of reactions in the biosynthetic pathway leading from pregnenolone to testosterone and several other steroid hormones. The first is the hydroxylation of pregnenolone or progesterone to the corresponding 17α-hydroxy steroid, followed by a lyase reaction that converts these 17α-hydroxy intermediates to the androgens dehydroepiandrosterone and androstenedione, respectively. cytochrome b5 (cytb5) is known to act as both an effector and electron donor for the lyase oxidations, markedly stimulating the rate of the lyase reaction in its presence relative to the rate in its absence. Extensive sequential backbone 1H,15N and 13C nuclear magnetic resonance assignments have now been made for oxidized CYP17A1 bound to the prostate cancer drug and inhibitor abiraterone. This is the first eukaryotic P450 for which such assignments are now available. These assignments allow more complete interpretation of the structural perturbations observed upon cytb5 addition. Possible mechanism(s) for the effector activity of cytb5 are discussed in light of this new information.

Novel Cytochrome P450 2C119 Enzymes in Cynomolgus and Rhesus Macaques Metabolize Progesterone, Diclofenac, and Omeprazole.

Cynomolgus and rhesus macaques are used in drug metabolism studies due to their evolutionary and phylogenetic closeness to humans. Cytochromes P450 (P450s or CYPs), including the CYP2C family enzyme, are important endogenous and exogenous substrate-metabolizing enzymes and play major roles in drug metabolism. In cynomolgus and rhesus macaques, six CYP2Cs have been identified and characterized, namely, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2C76, and CYP2C93. In this study, CYP2C119, a new CYP2C, was identified and characterized in cynomolgus and rhesus macaques. Cynomolgus and rhesus CYP2C119 contained open reading frames of 489 amino acids with high sequence identities to human CYP2C8 and to cynomolgus and rhesus CYP2C8. Phylogenetic analysis showed that cynomolgus and rhesus CYP2C119 were closely related to cynomolgus and rhesus CYP2C8. In cynomolgus and rhesus genomes, CYP2C genes, including CYP2C119, form a cluster. Among the tissues analyzed, cynomolgus CYP2C119 mRNA was predominantly expressed in liver. Hepatic expressions of CYP2C119 mRNA in four cynomolgus and two rhesus macaques varied, with no expression in one rhesus macaque. Among the CYP2C mRNAs, CYP2C119 mRNA was expressed less abundantly than CYP2C8, CYP2C9, CYP2C19, and CYP2C76 mRNAs but more abundantly than CYP2C18 mRNA. Recombinant cynomolgus and rhesus CYP2C119 catalyzed progesterone 16α-, 17α-, and 21-hydroxylation and diclofenac and omeprazole oxidations, indicating that CYP2C119 is a functional enzyme. Therefore, the novel CYP2C119 gene, expressed in macaque liver, encodes a functional enzyme that metabolizes human CYP2C substrates and is likely responsible for drug clearances. SIGNIFICANCE STATEMENT: Cytochrome P450 2C119 was found in cynomolgus and rhesus macaques, in addition to the known P450 2C8, 2C9, 2C18, 2C19, 2C76, and 2C93. Cynomolgus and rhesus CYP2C119 contain open reading frames of 489 amino acids with high sequence identity to human CYP2C8. Cynomolgus CYP2C119 mRNA is predominantly expressed in the liver. Recombinant CYP2C119 catalyzed progesterone hydroxylation and diclofenac and omeprazole oxidations. Therefore, the novel CYP2C119 gene expressed in the macaque liver encodes a functional enzyme that metabolizes human CYP2C substrates.

Crystal Structure and Biochemical Analysis of a Cytochrome P450 Steroid Hydroxylase (BaCYP106A6) from Bacillus Species.

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11β-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.

Rational design of thermophilic CYP119 for progesterone hydroxylation by in silico mutagenesis and docking screening

Steroid-based chemicals can affect the metabolism, immune functions, and development of sexual characteristics. Because of these effects, steroid derivatives are widely used in the pharmaceutical industry. Progesterone is a steroid-based hormone that mainly controls the ovulation period of women but is also a precursor molecule for the synthesis of important hormones like testosterone and cortisone. Cytochrome P450 (CYP) enzymes are important for the production of hydroxyprogesterones in the industry since they can catalyze regio- and enantioselective hydroxylation reactions. Although human CYP enzymes can catalyze hydroxyprogesterone synthesis with high selectivity, these enzymes are membrane bound, which limits their application for industrial production. CYP119 is a soluble and thermophilic enzyme from the archaea Sulfolobus acidocaldarius. Even though the native substrate of the enzyme is not known, CYP119 can catalyze styrene epoxidation, lauric acid hydroxylation, and Amplex®Red peroxidation. In this work, an in silico mutagenesis approach was used to design CYP119 mutants with high progesterone affinity. Energy scores of progesterone docking simulations were used for the design and elimination of single, double, and triple mutants of CYP119. Among designed 674 mutants, five of them match the criteria for progesterone hydroxylation. The most common mutation of these five mutants, L69G mutant was analyzed using independent molecular dynamics (MD) simulations in comparison with the wild-type (WT) enzyme. L69G CYP119, was expressed and isolated from Escherichia coli; it showed 800-fold higher affinity for progesterone compared to WT CYP119. L69G CYP119 also catalyzed progesterone hydroxylation. The novel designed enzyme L69G CYP119 is a potential versatile biocatalyst for progesterone hydroxylation that is expected to be stable under industrial production conditions.

Follicle isolation methods reveal plasticity of granulosa cell steroidogenic capacity during mouse in vitro follicle growth.

Follicles are the functional unit of the ovary and several methods have been developed to grow follicles ex vivo, which recapitulate key events of oogenesis and folliculogenesis. Enzymatic digestion protocols are often used to increase the yield of follicles from the ovary. However, the impact of these protocols on the outermost theca and granulosa cells, and thereby follicle function, is not well defined. To investigate the impact of enzymatic digestion on follicle function, we collected preantral follicles from CD1 mice either by enzymatic digestion (Enzy-FL) or mechanical isolation (Mech-FL) and compared follicle growth, steroidogenesis and cell differentiation within an encapsulated in vitro follicle growth system which maintains the 3D architecture of the oocyte and its surrounding somatic cells. Follicles were encapsulated in 0.5% alginate and cultured for 8 days. Compared with Enzy-FL, Mech-FL grew more rapidly and produced significantly higher levels of androstenedione, estradiol and progesterone. The expression of theca-interstitial cell marker genes, Cyp17a1, which encodes 17-hydroxylase/17, 20-lyase and catalyzes the hydroxylation of pregnenolone and progesterone to 17-hydroxypregnenolone and 17-hydroxyprogesterone, and the conversion of these products into dehydroepiandrosterone and androstenedione, and Star, which encodes a transport protein essential for cholesterol entry into mitochondria, were also higher in Mech-FL than in Enzy-FL. Mech-FL maintained an intact theca-interstitial layer on the outer edge of the follicle that phenocopied in vivo patterns as confirmed by alkaline phosphatase staining, whereas theca-interstitial cells were absent from Enzy-FL from the onset of culture. Therefore, preservation of the theca cell layer at the onset of culture better supports follicle growth and function. Interestingly, granulosa cells in the outermost layers of Enzy-FL expressed CYP17A1 by Day 4 of culture while maintaining inhibin α-subunit expression and a cuboidal nucleus. Thus, in the absence of theca-interstitial cells, granulosa cells have the potential to differentiate into androgen-producing cells. This work may have implications for human follicle culture, where enzymatic isolation is required owing to the density of the ovarian cortex.

Effect of Human Cytochrome P450 2D6 Polymorphism on Progesterone Hydroxylation.

Herein, hydroxylation activities at the 6β-position and 21-position of progesterone mediated by human cytochrome P450 (CYP) 2D6 and its variants and the effects of psychotropic drugs on these hydroxylation activities were compared to clarify whether CYP2D6 polymorphisms and psychotropic drugs impact neurosteroid levels in the brain. Progesterone was incubated with CYP2D6.1, CYP2D6.2 (Arg296Cys, Ser486Thr), CYP2D6.10 (Pro34Ser, Ser486Thr), and CYP2D6.39 (Ser486Thr) in the absence or presence of typical psychotropic drugs (fluvoxamine, fluoxetine, paroxetine, fluphenazine, and milnacipran) and endogenous steroids (testosterone and cortisol). Then, 6β- and 21-hydroxyprogesterone levels were determined by high-performance liquid chromatography. Although the Michaelis-Menten constants (Km) for progesterone 6β- and 21-hydroxylation reactions mediated by the different CYP2D6 variants were similar, the maximal velocity (Vmax) values of the reactions mediated by CYP2D6.1 and CYP2D6.2 were the highest, followed by those mediated by CYP2D6.39 and CYP2D6.10. Thus, the of progesterone 6β- and/or 21-hydroxylation reactions mediated by CYP2D6.1 and CYP2D6.2 showed the highest Vmax/Km values, followed by the reactions mediated by CYP2D6.39. All investigated compounds inhibited progesterone 21-hydroxylation mediated by CYP2D6 variants at high concentrations. Interestingly, at low concentrations, fluoxetine increased progesterone 21-hydroxylation mediated by CYP2D6.1, but not that mediated by CYP2D6.2 or CYP2D6.10. In addition, the Km value for CYP2D6.2 was elevated in the presence of fluoxetine, whereas the value for CYP2D6.1 was unaltered; however, Vmax values of both CYP2D6.1 and CYP2D6.2 were increased. Paroxetine competitively inhibited CYP2D6.1- and CYP2D6.2-mediated progesterone 21-hydroxylation. These results suggest that CYP2D6 polymorphism can affect the stimulation/inhibition of progesterone 21-hydroxylation.

Hydroxylation of Progesterone and Its Derivatives by the Entomopathogenic Strain Isaria farinosa KCh KW1.1.

Progesterone biotransformation is worth studying because of the high industrial value of its derivatives. This study investigated the catalytic ability of the entomopathogenic filamentous fungus strain Isaria farinosa KCh KW1.1 to transform progesterone derivatives: 11α-hydroxyprogesterone, 17α-hydroxyprogesterone, 16α,17α-epoxyprogesterone and pregnenolone. In the culture of Isaria farinosa KCh KW1.1, 11α-hydroxyprogesterone was effectively transformed into only one product: 6β,11α-dihydroxyprogesterone. Transformation of 17α-hydroxyprogesterone gave three hydroxy derivatives: 6β,17α-dihydroxyprogesterone, 12β,17α-dihydroxyprogesterone and 6β,12β,17α-trihydroxyprogesterone. Two products: 6β-hydroxy-16α,17α-epoxyprogesterone and 6β,11α-dihydroxy-16α,17α-epoxyprogesterone, were obtained from the 16α,17α-epoxyprogesterone transformation. We isolated two compounds from the biotransformation medium with pregnenolone: 11α-hydroxy-7-oxopregnenolone and 5α,6α-epoxy-3β,11α-dihydroxypregnan-7,20-dione. In this study, we observed only mono- and dihydroxy derivatives of the tested substrates, and the number of obtained products for each biotransformation did not exceed three.

CYP21A2 Gene Expression in a Humanized 21-Hydroxylase Mouse Model Does Not Affect Adrenocortical Morphology and Function.

Steroid 21-hydroxylase is an enzyme of the steroid pathway that is involved in the biosynthesis of cortisol and aldosterone by hydroxylation of 17α-hydroxyprogesterone and progesterone at the C21 position. Mutations in CYP21A2, the gene encoding 21-hydroxylase, cause the most frequent form of the autosomal recessive disorder congenital adrenal hyperplasia (CAH). In this study, we generated a humanized 21-hydroxylase mouse model as the first step to the generation of mutant mice with different CAH-causing mutations. We replaced the mouse Cyp21a1 gene with the human CYP21A2 gene using homologous recombination in combination with CRISPR/Cas9 technique. The aim of this study was to characterize the new humanized mouse model. All results described are related to the homozygous animals in comparison with wild-type mice. We show analogous expression patterns of human 21-hydroxylase by the murine promoter and regulatory elements in comparison to murine 21-hydroxylase in wild-type animals. As expected, no Cyp21a1 transcript was detected in homozygous CYP21A2 adrenal glands. Alterations in adrenal gene expression were observed for Cyp11a1, Star, and Cyb11b1. These differences, however, were not pathological. Outward appearance, viability, growth, and fertility were not affected in the humanized CYP21A2 mice. Plasma steroid levels of corticosterone and aldosterone showed no pathological reduction. In addition, adrenal gland morphology and zonation were similar in both the humanized and the wild-type mice. In conclusion, humanized homozygous CYP21A2 mice developed normally and showed no differences in histological analyses, no reduction in adrenal and gonadal gene expression, or in plasma steroids in comparison with wild-type littermates.

Augmentation of progestin signaling rescues testis organization and spermatogenesis in zebrafish with the depletion of androgen signaling.

Disruption of androgen signaling is known to cause testicular malformation and defective spermatogenesis in zebrafish. However, knockout of cyp17a1, a key enzyme responsible for the androgen synthesis, in ar-/- male zebrafish paradoxically causes testicular hypertrophy and enhanced spermatogenesis. Because Cyp17a1 plays key roles in hydroxylation of pregnenolone and progesterone (P4), and converts 17α-hydroxypregnenolone to dehydroepiandrosterone and 17α-hydroxyprogesterone to androstenedione, we hypothesize that the unexpected phenotype in cyp17a1-/-;androgen receptor (ar)-/- zebrafish may be mediated through an augmentation of progestin/nuclear progestin receptor (nPgr) signaling. In support of this hypothesis, we show that knockout of cyp17a1 leads to accumulation of 17α,20β-dihydroxy-4-pregnen-3-one (DHP) and P4. Further, administration of progestin, a synthetic DHP mimetic, is sufficient to rescue testicular development and spermatogenesis in ar-/- zebrafish, whereas knockout of npgr abolishes the rescue effect of cyp17a1-/- in the cyp17a1-/-;ar-/- double mutant. Analyses of the transcriptomes among the mutants with defective testicular organization and spermatogenesis (ar-/-, ar-/-;npgr-/- and cyp17a-/-;ar-/-;npgr-/-), those with normal phenotype (control and cyp17a1-/-), and rescued phenotype (cyp17a1-/-;ar-/-) reveal a common link between a downregulated expression of insl3 and its related downstream genes in cyp17a-/-;ar-/-;npgr-/- zebrafish. Taken together, our data suggest that genetic or pharmacological augmentation of the progestin/nPgr pathway is sufficient to restore testis organization and spermatogenesis in zebrafish with the depletion of androgen signaling.

Contribution of the human cytochrome P450 2C subfamily to the metabolism of and the interactions with endogenous compounds including steroid hormones.

The objectives of this study were as follows: 1) to compare the metabolic activities of endogenous compounds and their effects on dopamine formation and hydroxylation of steroid hormones, mediated by human cytochrome P450 (CYP), including CYP2C9.1 and CYP2C19, as well as the variants CYP2C9.2 (Arg144Cys) and CYP2C9.3 (Ile359Leu); and 2) to assess the effects of steroid hormones on the activities of CYP2C9.1, CYP2C9.2, and CYP2C19 to estimate the contribution of the CYP2C subfamily to metabolism and drug-drug interactions of endogenous compounds. Dopamine formation from p -tyramine and 6β- and 21- (for progesterone) hydroxylation of testosterone, cortisol, and progesterone by CYP2C9 variants, CYP2C19, CYP2D6, and CYP3A4 were determined using HPLC. The effects of steroid hormones such as testosterone, cortisol, and progesterone on tolbutamide methyl hydroxylation mediated by CYP2C subfamily members were investigated. Only CYP2D6 catalyzed dopamine formation. The 6β-hydroxylation activities of testosterone, cortisol, and progesterone catalyzed by CYP2C9 variants and CYP2D6 were less than 5% of those by CYP3A4. Although cortisol did not inhibit tolbutamide methyl hydroxylation catalyzed by CYP2C9.1, CYP2C9.2, or CYP2C19 and testosterone did not inhibit CYP2C19 activity, the reactions catalyzed by CY2C9.1 and CYP2C9.2 were inhibited by testosterone. The inhibition of progesterone by CYP2C19 was stronger than that by CYP2C9.1 and CYP2C9.2. CYP2C9.1 and CYP2C19 noncompetitively and competitively inhibited tolbutamide methyl hydroxylation with inhibition constants of 43.2 μM and 1.03 μM, respectively. Clinical interactions among endogenous compounds would vary within the CYP2C subfamily, although the contribution of the CYP2C subfamily may be of minor importance for dopamine formation and the detoxification (6β-hydroxylation) of endogenous steroid hormones.

Identification of a novel cytochrome P450 17A2 enzyme catalyzing the C17α hydroxylation of progesterone and its application in engineered Pichia pastoris

The 17α-hydroxyprogesterone (17-HP) is an important steroid drug. Progesterone-17α-hydroxylase (CYP17A) transforms progesterone into 17-HP; however, the major limitation in the bioproduction of 17-HP is the lack of CYP17A2 or low activity of the existing enzymes. Herein, a novel CYP17A2 from Mastacembelus armatus (MA_CYP17A2) was obtained through genome mining and expressed in Pichia pastoris GS115. The biotransformation of 17-HP was performed in whole cells and the product analysis indicated that MA_CYP17A2 possessed the activities of progesterone C17-hydroxylate (17-HP titer, 43.5 ± 4.7 mg/L). Moreover, cytochrome P450 oxidoreductase (CPR) was optimized from five different species by co-expressing them in engineered GS115-MA_CYP17A2 with dual plasmids for boosting the electron-transfer. Furthermore, glucose-6-phosphate dehydrogenase (ZWF) was optimized from three different species and used for enhancing the regeneration of NADPH in the presence of glucose. After optimization, a binary vector of pPICZB-cpr-zwf containing optimal cprrat from Rattus norvegicus and zwfk from Kluyveromyces lactis was constructed and electro-transformed into GS115-MA_CYP17A2 for the co-expression of three foreign proteins in one host. Finally, the synthetic ability of 17-HP on GS115-MA_CYP17A2-CPRRAT-ZWFK was determined, the 17-HP titer improved by 2.8-fold and 17-HP specific productivity improved by 3.0-fold compared to GS115-MA_CYP17A2. The heterologous expression system GS115-MA_CYP17A2-CPRRAT-ZWFK has been reported to cause the highest 17-HP bioconversion. This study provides a promising hydroxylation system candidate for the efficient biosynthesis of 17-HP.

CYP11B and CYP17A1 in the Biosynthesis and Metabolism of C11‐oxy Steroids

Cytochrome P450 (CYP11B1) and cytochrome 17α‐hydroxylase (CYP17A1) play pivotal roles in the biosynthesis of adrenal glucocorticoids and androgens. It is generally accepted that CYP11B1 catalyses only the production of cortisol and corticosterone while CYP17A1 catalyses the hydroxylation of pregnenolone and progesterone at C17 and, in humans, the 17,20‐lyase of 17‐hydroxypregnenolone to produce DHEA. CYP11B1, however, also catalyses the biosynthesis of C11‐oxy C21 steroids, 11β‐hydroxyandrostenedione, 11β‐hydroxyprogesterone (11OHPROG) and 21‐deoxycortisol (21dF) in the adrenal [1, 2].We investigated the potential role of CYP17A1 in the metabolism of these C11‐oxy C21steroids leading to the production of 11keto‐dihydrotestosterone since 17‐hydroxyprogesterone has been shown to lead to the production of dihydrotestosterone in the backdoor pathway.The 17α‐hydroxylase/17,20‐lyase activity of CYP17A1 towards 11OHPROG and 21dF was assayed as well as the catalytic activity of the enzyme towards the downstream C11‐oxy C21steroid products. In vitro conversions were carried out with 1 μM steroid substrate in HEK293 and in LNCaP cells transiently expressing CYP17A1 and metabolites analysed using UPC2‐MS/MS. Both 11OHPROG and 11keto‐progesterone (11KPROG) were hydroxylated at C17 producing 21dF, 0.27 μM and 21‐deoxycortisone, 0.65 μM, respectively while no lyase activity was detected. It was only once reduced by 5α‐reductase and 3α‐hydroxysteroid dehydrogenase that the aforementioned steroids were also lyased to produce the C11‐oxy C19steroids, 11‐hydroxyandrosterone, 0.6 μM and 11‐ketoandrosterone, 0.43 μM. In LnCaP cells 11OHPROG and 11KPROG were metabolised to C11‐oxy C19steroids indicative of the C11‐oxy backdoor pathway. In addition, in HEK293 cells transiently transfected with 11β‐hydroxysteroid dehydrogenase (11βHSD) type 1 and 11βHSD2 the isozymes readily catalysed the interconversion of all the C11‐keto C21steroid intermediates produced in the pathway with the production of the keto intermediates being favoured.Although the C11‐oxy C21steroids produced in the adrenal may serve as ligands for the progesterone receptor they also potentially add to the pool of potent androgens via the C11‐oxy backdoor pathway. These C11‐oxy steroids would thus further contribute to the androgen pool in adrenal disorders and diseases characterised by androgen excess in which these steroids have been identified. We recently reported 11OHPROG, 11keto‐dihydroprogesterone and pregnanetriol in circulation in BPH patients [3]. The C11‐oxy C21steroids and C11‐oxy C19steroids, however, remain to be fully explored –not only in pathophysiology but also in sexual differentiation and development.Support or Funding InformationThe South African National Research Foundation (IFR170125217588, CSUR160414162143)

Novel approach to improve progesterone hydroxylation selectivity by CYP106A2 via rational design of adrenodoxin binding.

Bacterial P450s have considerable potential for biotechnological applications. The P450 CYP106A2 from Bacillus megaterium ATCC 13368 converts progesterone to several hydroxylated products that are important precursors for pharmaceutical substances. As high yields of monohydroxylated products are required for biotechnological processes, improving this conversion is of considerable interest. It has previously been shown that the binding mode of the redox partner can affect the selectivity of the progesterone hydroxylation, being more stringent in case of the Etp1 compared with Adx(4-108). Therefore, in this study we aimed to improve hydroxylation selectivity by optimizing the binding of Adx(4-108) with CYP106A2 allowing for a shorter distance between both redox centers. To change the putative binding interface of Adx(4-108) with CYP106A2, molecular docking was used to choose mutation sites for alteration. Mutants at positions Y82 and P108 of Adx were produced and investigated, and confirmed our hypothesis. Protein-protein docking, as well as conversion studies, using the mutants demonstrated that the iron-sulfur(FeS) cluster/heme distance diminished significantly, which subsequently led to an approximately 2.5-fold increase in 15β-hydroxyprogesterone, the main product of progesterone conversion by CYP106A2.

Binding modes of CYP106A2 redox partners determine differences in progesterone hydroxylation product patterns

Natural redox partners of bacterial cytochrome P450s (P450s) are mostly unknown. Therefore, substrate conversions are performed with heterologous redox partners; in the case of CYP106A2 from Bacillus megaterium ATCC 13368, bovine adrenodoxin (Adx) and adrenodoxin reductase (AdR). Our aim was to optimize the redox system for CYP106A2 for improved product formation by testing 11 different combinations of redox partners. We found that electron transfer protein 1(516–618) showed the highest yield of the main product, 15β-hydroxyprogesterone, and, furthermore, produced a reduced amount of unwanted polyhydroxylated side products. Molecular protein–protein docking indicated that this is caused by subtle structural changes leading to alternative binding modes of both redox enzymes. Stopped-flow measurements analyzing the CYP106A2 reduction and showing substantial differences in the apparent rate constants supported this conclusion. The study provides for the first time to our knowledge rational explanations for differences in product patterns of a cytochrome P450 caused by difference in the binding mode of the redox partners.

Structure-Based Engineering of Steroidogenic CYP260A1 for Stereo- and Regioselective Hydroxylation of Progesterone

The production of regio- and stereoselectively hydroxylated steroids is of high pharmaceutical interest and can be achieved by cytochrome P450-based biocatalysts. CYP260A1 from Sorangium cellulosum strain So ce56 catalyzes hydroxylation of C19 or C21 steroids at the very unique 1α-position. However, the conversion of progesterone (PROG) by CYP260A1 is very unselective. In order to improve its selectivity we applied a semirational protein engineering approach, resulting in two different, highly regio- and stereoselective mutants by replacing a single serine residue (S276) of the substrate recognition site 5 with an asparagine or isoleucine. The S276N mutant converted PROG predominantly into 1α-hydroxy-PROG, while the S276I mutant led to 17α-hydroxy-PROG. We solved the high-resolution crystal structures of the PROG-bound S276N and S276I mutants, which revealed two different binding modes of PROG in the active site. The orientations were consistent with the exclusive 1α- (pro-1α binding mode) and 17α-hydroxylation (pro-17α-binding mode) of S276N and S276I, respectively. We observed that water-mediated hydrogen bonds contribute to the stabilization of the polar C3 and C17 substituents of PROG. Both binding modes of PROG may be stabilized in the wild-type enzyme. The change in regioselectivity is mainly driven by destabilizing the alternative binding mode due to steric hindrance and hydrogen bond disruption, caused by the mutations of Ser276. Thus, for the first time, the change in the selectivity of cytochrome P450-mediated steroid hydroxylation created by rational mutagenesis can be explained by the obtained 3D structures of the substrate-bound mutants, providing the basis for further experiments to engineer the biocatalyst toward novel steroid hydroxylation positions.

Progesterone hydroxylation by cytochromes P450 2C and 3A enzymes in marmoset liver microsomes

1. Common marmosets (Callithrix jacchus) are potentially useful nonhuman primate models for preclinical drug metabolism studies. However, the roles of marmoset cytochrome P450 (P450) isoforms in the oxidation of endobiotic progesterone have not been fully investigated. In this study, the roles of marmoset P450 isoforms in progesterone hydroxylation were extensively determined.2. The activities of liver microsomes from individual marmosets with respect to progesterone 21/17α- and 16α/6β-hydroxylation were significantly correlated with those for flurbiprofen 4-hydroxylation and midazolam 1′-hydroxylation, respectively, as similar correlations have been found in humans. Anti-P450 2 C and 3 A antibodies suppressed progesterone 21/17α- and 16α/6β-hydroxylation, respectively, in marmoset liver microsomes.3. Recombinant marmoset P450 2C58 and 2C19 catalyzed progesterone to form 21-hydroxyprogesterone and 16α-hydroxyprogesterone, respectively, as major products with high maximum velocity/Km values of 0.53 and 0.089 mL/min/nmol, respectively. Recombinant marmoset P450 3A4/90 oxidized progesterone to form 6β-hydroxyprogesterone as a major product with homotropic cooperativity (>1 of Hill coefficients).4. These results indicate that the overall activities and roles of liver microsomal P450 enzymes in marmoset livers are similar to those in humans, especially for progesterone 21/17α- and 16α/6β-hydroxylation by marmoset P450 2 C and 3 A enzymes, respectively, suggesting important roles for these P450 enzymes in the metabolism of endobiotics in marmosets.