Molecular basis of 17α-hydroxylase/17,20-lyase deficiency

17α-Hydroxylase deficiency is characterized by a defect in either or both of 17α-hydroxylase and 17,20-lyase activities, based on the fact that a single polypeptide P450c17 can catalyze both reactions. The clinical manifestations of 17α-hydroxylase/17,20-lyase deficiency seem to be more heterogeneous than expected, varying from the classical type to less symptomatic forms as also observed in 21-hydroxylase deficiency. We have sequenced all eight exons of the CYP17 (P450c17) gene in DNA from several patients, reconstructed the mutations in a human P450c17 cDNA and expressed the mutant P450c17 in COS 1 cells to characterize the kinetic properties of 17α-hydroxylase and 17,20-lyase activities. The molecular bases of cases clinically reported as 17α-hydroxylase deficiency have turned out to be complete or partial combined deficiencies of 17α-hydroxylase/17,20-lyase. The elucidation of the molecular basis generally explains the patient's clinical profiles including the sexual phenotype of the external genitalia. In one case clinically reported as isolated 17,20-lyase deficiency, the molecular basis was found to be partial combined deficiency of both activities, somewhat discordant with the patient's clinical profile. Based on the results obtained so far we can predict that those 17α-hydroxylase deficient individuals having a homozygous stop codon in the CYP17 gene positioned at the amino terminal side of the P450c17 heme-binding cysteine (442) will all have the same phenotype. However those individuals having homozygous missense mutations or those who are compound heterozygotes having a missense mutation in at least one CYP17 allele will display their own unique phenotype which clinically will be subtly different from all others.     

An alternative explanation of hypertension associated with 17α-hydroxylase deficiency syndrome

The syndrome of 17α-hydroxylase deficiency is due to the inability to synthesize cortisol and is associated with enhanced secretion of both corticosterone and 11-deoxy-corticosterone (DOC). In humans, corticosterone and its 5α-Ring A-reduced metabolites are excreted via the bile into the intestine and transformed by anaerobic bacteria to 21-dehydroxylated products: 11β-OH-progesterone or 11β-OH-(allo)-5α-preganolones (potent inhibitors of 11β-HSD2 and 11β-HSD1 dehydrogenase). Neomycin blocks the formation of these steroid metabolites and can blunt the hypertension in rats induced by either ACTH or corticosterone. 3α,5α-Tetrahydro-corticosterone, 11β-hydroxy-progesterone, and 3α,5α-tetrahydro-11β-hydroxy-progesterone strongly inhibit 11β-HSD2 and 11β-HSD1 dehydrogenase activity; all these compounds are hypertensinogenic when infused in adrenally intact rats.Urine obtained from a patient with 17α-hydroxylase deficiency demonstrated markedly elevated levels of endogenous glycyrrhetinic acid-like factors (GALFs) that inhibit 11β-HSD2 and 11β-HSD1 dehydrogenase activity (>300 times greater, and >400 times greater, respectively, than those in normotensive controls). Thus, in addition to DOC, corticosterone and its 5α-pathway products as well as the 11-oxygenated progesterone derivatives may play a previously unrecognized role in the increased Na⁺ retention and BP associated with patients with 17α-hydroxylase deficiency.     

Structural characterization of human cholesterol 7α-hydroxylase

Hepatic conversion to bile acids is a major elimination route for cholesterol in mammals. CYP7A1 catalyzes the first and rate-limiting step in classic bile acid biosynthesis, converting cholesterol to 7α-hydroxycholesterol. To identify the structural determinants that govern the stereospecific hydroxylation of cholesterol, we solved the crystal structure of CYP7A1 in the ligand-free state. The structure-based mutation T104L in the B′ helix, corresponding to the nonpolar residue of CYP7B1, was used to obtain crystals of complexes with cholest-4-en-3-one and with cholesterol oxidation product 7-ketocholesterol (7KCh). The structures reveal a motif of residues that promote cholest-4-en-3-one binding parallel to the heme, thus positioning the C7 atom for hydroxylation. Additional regions of the binding cavity (most distant from the access channel) are involved to accommodate the elongated conformation of the aliphatic side chain. Structural complex with 7KCh shows an active site rigidity and provides an explanation for its inhibitory effect. Based on our previously published data, we proposed a model of cholesterol abstraction from the membrane by CYP7A1 for metabolism. CYP7A1 structural data provide a molecular basis for understanding of the diversity of 7α-hydroxylases, on the one hand, and cholesterol-metabolizing enzymes adapted for their specific activity, on the other hand.     

Identifying a novel mutation of CYP17A1 gene from five Chinese 17α-hydroxylase/17, 20-lyase deficiency patients

Mutations of CYP17A1 gene could cause complete or partial, combined or isolated 17α-hydroxylase/17,20-lyase enzyme deficiencies (17OHD). We intended to investigate the CYP17A1 mutation in five unrelated patients and analyze its possible influence on phenotype of an atypical 17OHD patient presented with micropenis, hypertension and intermittent hypokalemia. Steroid hormones were assayed in these patients. A novel missense mutation (c.1169C>G, p. Thr390Arg) located in exon 7 was detected in one of the patients. Homozygous c. 985_987delinsAA, p. Tyr329fs mutation was found in two patients, while compound heterozygous mutations (c. 985_987delinsAA, p. Tyr329fs/c. 932–939 del, p. Val311fs and c. 287G>A, p. Arg96Gln/c. 985_987delinsAA, p. Tyr329fs) were found in two other patients, respectively. Then, steric model analysis of CYP17A1 showed that the novel mutation T390R changed the local structure as well as the electrostatic potential of the nearby beta sheet. Finally, site-directed mutagenesis and in vitro expression were used to analyze the activity of novel mutant CYP17A1. It indicated the T390R mutant retained part of enzyme activity, which was consistent to the clinical features. In conclusion, we identified a novel missense mutation of CYP17A1 gene from a patient with micropenis, hypertension and intermittent hypokalemia, which varied from other four patients. It also expanded our understanding of genotype–phenotype correlation of the disease.     

Regeneration of the progesterone 11α-hydroxylase of Rhizopus nigricans by the use of periodate

Summary The cell-free progesterone 11-hydroxylase enzyme of Rhizopus nigricans can be directly regenerated by periodate oxidation. This permits action of the enzyme over a period of hours with an activity similar to that in the presence of an NADPH generating system.     

Novel series of 17β-pyrazolylandrosta-5,16-diene derivatives and their inhibitory effect on 17α-hydroxylase/C(17,20)-lyase

The Claisen condensation of 3β-acetoxypregna-5,16-dien-20-one (1) with ethyl formate in the presence of sodium methylate in pyridine is known to lead to 3β-hydroxy-21-hydroxymethylidenepregna-5,16-dien-20-one (2) in good yield. With the methods described for the preparation of the saturated D-ring pyrazolyl series, the reactions of 2 with phenylhydrazine and its p-substituted derivatives in acetic acid resulted in mixtures of two steroidal regioisomers, the 1'-aryl-3'-pyrazolyl-(4a-e) and 1'-aryl-5'-pyrazolyl (5a-e) steroids. Compounds 4a-e are unknown in the literature. The arylpyrazoles produced were tested against 17α-hydroxylase/C(17,20)-lyase (P450(17α)) in vitro and neither of the regioisomers exerted efficient inhibition.     

Identification of a common molecular basis for combined 17α-hydroxylase/17,20-lyase deficiency in two Mennonite families

Summary During the course of studies to characterize mutations of the CYP17 gene that cause the 17-hydroxylase-deficient form of congenital adrenal hyperplasia we have discovered two ostensibly unrelated Mennonite families in which affected individuals are homozygous for the same mutation. The defect is a four-base duplication in exon 8 of the CYP17 gene, which alters the reading frame encoding the C-terminal 26 animo acids of cytochrome P450₁₇.     

Canadian Mennonites and individuals residing in the Friesland region of the Netherlands share the same molecular basis of 17α-hydroxylase deficiency

Summary A common mutation within the CYP17 gene that causes 17-hydroxylase deficiency, a form of congenital adrenal hyperplasia, has been found by direct sequencing of polymerase chain reaction (PCR) fragments of genomic DNA from six families residing in the Friesland region of the Netherlands. The mutation is a 4-base duplication within exon 8 of the CYP17 gene, which alters the reading frame encoding the C-terminal 26 amino acids of cytochrome P45017. This mutation has previously been found in two Canadian patients who are members of ostensibly unrelated Mennonite families. The Mennonite Churches derive their name from Menno Simons, an early leader of the sect in Friesland. Presumably this 4-base duplication appeared within the Friesian population prior to emigration of the Mennonites from the Netherlands.     

Synthesis of regioisomeric 17β-N-phenylpyrazolyl steroid derivatives and their inhibitory effect on 17α-hydroxylase/C17,20-lyase

The reaction of 3β-hydroxy-21-hydroxymethylidenepregn-5-en-3β-ol-20-one (1) with phenylhydrazine (2a) affords two regioisomers, 17β-(1-phenyl-3-pyrazolyl)androst-3-en-3β-ol (5a) and 17β-(1-phenyl-5-pyrazolyl)androst-5-en-3β-ol (6a). The direction of the ring-closure reactions of 1 with p-substituted phenylhydrazines (2b-e) depends strongly on the electronic features of the substituents. Oppenauer oxidation of 3β-hydroxy-17β-exo-heterocyclic steroids 5a-e and 6a-e yielded the corresponding Δ⁴-3-ketosteroids 9a-e and 10a-e. The inhibitory effects (IC₅₀) of these compounds on rat testicular C_17,20-lyase were investigated by means of an in vitro radioligand incubation technique.     

Novel oxazolinyl derivatives of pregna-5,17(20)-diene as 17α-hydroxylase/17,20-lyase (CYP17A1) inhibitors

New oxazolinyl derivatives of [17(20)E]-pregna-5,17(20)-diene: 2′-{[(E)-3β-hydroxyandrost-5-en-17-ylidene]methyl}-4′,5′-dihydro-1′,3′-oxazole 1 and 2′-{[(E)-3β-hydroxyandrost-5-en-17-ylidene]methyl}-4′,4′-dimethyl-4′,5′-dihydro-1′,3′-oxazole 2 were evaluated as potential CYP17A1 inhibitors in comparison with 17-(pyridin-3-yl)androsta-5,16-dien-3β-ol 3 (abiraterone). Differential absorption spectra of human recombinant CYP17A1 in the presence of compound 1 (λ_max = 422 nm, λ_min = 386 nm) and compound 2 (λ_max = 416 nm) indicated significant differences in enzyme/inhibitors complexes. CYP17A1 activity was measured using electrochemical methods. Inhibitory activity of compound 1 was comparable with abiraterone 3 (IC₅₀ = 0.9 ± 0.1 μM, and IC₅₀ = 1.3 ± 0.1 μM, for compounds 1 and 3, respectively), while compound 2 was found to be weaker inhibitor (IC₅₀ = 13 ± 1 μM). Docking of aforementioned compounds to CYP17A1 revealed that steroid fragments of compound 1 and abiraterone 3 occupied close positions; oxazoline cycle of compound 1 was coordinated with heme iron similarly to pyridine cycle of abiraterone 3. Configuration of substituents at 17(20) double bond in preferred docked position corresponded to Z-isomers of compounds 1 and 2. Presence of 4′-substituents in oxazoline ring of compound 2 prevents coordination of oxazoline nitrogen with heme iron and worsens its docking score in comparison with compound 1. These data indicate that oxazolinyl derivative of [17(20)E]-pregna-5,17(20)-diene 1 (rather than 4′,4′-dimethyl derivative 2) may be considered as potential CYP17A1 inhibitor and template for development of new compounds affecting growth and proliferation of prostate cancer cells.     

Synthesis and biochemical evaluation of a range of sulfonated derivatives of 4-hydroxybenzyl imidazole as highly potent inhibitors of rat testicular 17α-hydroxylase/17,20-lyase (P-45017α)

We report the synthesis and biochemical evaluation of a range of 4-sulfonated derivatives of 4-hydroxybenzyl imidazole which has been targetted against the two components of 17α-hydroxylase/17,20-lyase (P-450_17α), namely, 17α-hydroxylase (17α-OHase) and 17,20-lyase (lyase). The results from the biochemical testing suggest that the compounds synthesised are highly potent inhibitors possessing excellent selectivity towards the lyase component.     

Synthesis of 16α-3H androgen and estrogen substrates for 16α-hydroxylase

The synthesis of 16α-³H androgens and estrogens is described. 1-(³H)-Acetic acid in the presence of zinc dust reacts with 16α-bromo-17-ketosteroids to produce 16α-³H-17-ketosteroids. This chemical reaction was used to prepare 16α-³H-dehydroepiandrosterone (I) and 16α-³H-estrone acetate (XI) from 16α-bromo-dehydroepiandrosterone (X) and from 16α-bromo-estrone acetate (XII), respectively. Using appropriate microbiological techniques, it was possible to convert these radiolabelled substrates into 16α-³H-androstenedione (II) and 16α-³H-estradiol-17β (VII). 16α-³H-Estrone (VI) was obtained by the chemical hydrolysis of 16α-³H-estrone acetate. The label distribution as determined by microbiological 16α-hydroxylations indicated a specific labelling of 77% for androgens and 65% for estrogens in the 16α position. These substrates can be used for measuring the 16α hydroxylase activity, an important step in the biosynthesis of estriol (VIII) and estetrol (IX).     

Metabolism of 17α-methyl-5α-dihydrotestosterone in the rabbit

17α-Methyl-5α-dihydrotestosterone and the reduced metabolites, 17α-methyl-5α-androstane-3α, 17β-diol and -3β, 17β-diol together with two hydroxylated metabolites, 17α-methyl-5α-androstane-3β, 15α, 17β-triol and 17α-methyl-5α-androstane-3α, 6α, 17β-triol were isolated and identified in the urine of rabbits orally dosed with 17α-methyl-5α-dihydrotestosterone. Formation of the C-6 hydroxylated derivative demonstrates that the 4,6-enolization of a 4-en-3-one is not a necessary requirement for hydroxylation at C-6 of the androstane nucleus in the rabbit. No evidence was obtained for the presence of 17α-methyl/17β-hydroxyl epimerization.     

Photochemical action spectrum of the co-inhibited 5β-cholestane-3α, 7α, 12α-triol 26-hydroxylase system

The inhibition of the mitochondrial hydroxylation of 5β-cholestane-3α, 7α, 12α-triol at the 26 position by a CO:O₂ gas mixture was maximally reversed by monochromatic light at the wavelength of 450 nm. This establishes the involvement of a cytochrome P450 dependent monooxygenase in the 26-hydroxylation of 5β-cholestane-3α, 7α, 12α-triol in rat liver mitochondria.     

Cholesterol 7α-hydroxylase in rat liver microsomal preparations

Subcellular fractions containing microsomes prepared from rat livers homogenized in the absence of EDTA catalysed the oxidation of cholesterol to 7alpha-hydroxycholesterol, 7-oxocholesterol, 7beta-hydroxycholesterol and 5alpha-cholestane-3beta,5,6beta-triol. These reactions required native protein, molecular oxygen and NADPH. It is suggested that these compounds are formed by a peroxidation analogous to the peroxidation of fatty acids catalysed by liver microsomal preparations. Incubations of [4-(14)C]cholesterol with microsomal preparations from rat liver homogenized in the presence of EDTA gave 7alpha-hydroxy[(14)C]cholesterol as the main product. This reaction required molecular oxygen and NADPH, and was inhibited by CO. The mass of 7alpha-hydroxycholesterol formed during the incubation was measured by a double-isotope-derivative dilution procedure. This procedure was used to assay the activity of cholesterol 7alpha-hydroxylase and to measure low concentrations of endogenous 7alpha-hydroxycholesterol in liver.     

Screening of mycelial fungi for 7α- and 7β-hydroxylase activity towards dehydroepiandrosterone

In total, 481 fungal strains were screened for the ability to carry out 7(α/β)-hydroxylation of dehydroepiandrosterone (DHEA, 3β-hydroxy-5-androsten-17-one). Representatives of 31 genera of 15 families and nine orders of ascomycetes, 17 genera of nine families and two orders of zygomycetes, two genera of two families and two orders of basidiomycetes, and 14 genera of mitosporic fungi expressed 7(α/β)-hydroxylase activity. The majority of strains were able to introduce a hydroxyl group to position 7α. Active strains selectively producing 3β,7α-dihydroxy-5-androsten-17-one were found among Actinomucor, Backusella, Benjaminiella, Epicoccum, Fusarium, Phycomyces and Trichothecium, with the highest yield of 1.25 and 1.9 g L^?1 from 2 and 5 g L^?1 DHEA, respectively, reached with F. oxysporum. Representatives of Acremonium, Bipolaris, Conidiobolus and Curvularia formed 3β,7β-dihydroxy-5-androsten-17-one as a major product from DHEA. The structures of the major steroid products were confirmed by TLC, gas chromatography (GC), mass spectra (MS), and ¹H-NMR analyses.     

Inhibition of dopamine-β-hydroxylase by cytokinins

The activity of cell-free preparations of dopamine--hydroxylase from a mammalian source was inhibited by a number of N⁶-substituted adenine derivatives that are hormonally active as cytokinins in plant systems. The synthetic cytokinin N⁶-cyclohexylmethyladenine exhibited inhibitory activity equivalent to that of 1-phenyl-3-(2-thiazolyl)-2-thiourea (PTTU), a compound known to be a potent inhibitor of dopamine--hydroxylase activity. PTTU itself was found to exhibit cytokinin activity in the tobacco callus bioassay and to inhibit the activity of the plant enzyme, cytokinin oxidase. The possible significance of these observations is discussed in relation to known effects of cytokinins on phenethylamine metabolism.