Diversity and function of mutations in p450 oxidoreductase in patients with Antley-Bixler syndrome and disordered steroidogenesis

P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes. Although mouse Por gene ablation causes embryonic lethality, POR missense mutations cause disordered steroidogenesis, ambiguous genitalia, and Antley-Bixler syndrome (ABS), which has also been attributed to fibroblast growth factor receptor 2 (FGFR2) mutations. We sequenced the POR gene and FGFR2 exons 8 and 10 in 32 individuals with ABS and/or hormonal findings that suggested POR deficiency. POR and FGFR2 mutations segregated completely. Fifteen patients carried POR mutations on both alleles, 4 carried mutations on only one allele, 10 carried FGFR2 or FGFR3 mutations, and 3 patients carried no mutations. The 34 affected POR alleles included 10 with A287P (all from whites) and 7 with R457H (four Japanese, one African, two whites); 17 of the 34 alleles carried 16 "private" mutations, including 9 missense and 7 frameshift mutations. These 11 missense mutations, plus 10 others found in databases or reported elsewhere, were recreated by site-directed mutagenesis and were assessed by four assays: reduction of cytochrome c, oxidation of NADPH, support of 17alpha-hydroxylase activity, and support of 17,20 lyase using human P450c17. Assays that were based on cytochrome c, which is not a physiologic substrate for POR, correlated poorly with clinical phenotype, but assays that were based on POR's support of catalysis by P450c17--the enzyme most closely associated with the hormonal phenotype--provided an excellent genotype/phenotype correlation. Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency.     

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.     

Human cytochrome b5 requires residues E48 and E49 to stimulate the 17,20-lyase activity of cytochrome P450c17

Cytochrome P450c17 (CYP17) catalyzes both the 17alpha-hydroxylase and 17,20-lyase reactions in human steroid biosynthesis. Cytochrome b5 (b5) stimulates the rate of the 17,20-lyase reaction 10-fold with little influence on 17alpha-hydroxylase activity. Studies with apo-b5 suggest that stimulation of 17,20-lyase activity results from an allosteric action on the hCYP17 x POR complex, rather than electron transfer by b5. We hypothesized that specific residues on b5 interact with the hCYP17 x POR complex and that targeted mutation of surface-exposed residues might identify b5 residues critical for stimulating 17,20-lyase activity. We constructed, expressed, and purified 14 single plus 3 double b5 mutations and assayed their ability to stimulate 17,20-lyase activity. Most mutations did not alter the capacity of b5 to stimulate 17,20-lyase activity or appeared to modestly alter the affinity of b5 for the hCYP17 x POR complex. In contrast, mutation of E48, E49, or R52 reduced the maximal stimulation of 17,20-lyase activity. In particular, b5 mutation E48G + E49G lost over 95% of the capacity to stimulate 17,20-lyase activity, yet this mutation retained normal electron transfer properties. In addition, mutation E48G + E49G did not impair stimulation of 17,20-lyase activity by wild-type b5, suggesting that the mutation binds poorly to the site of the hCYP17 x POR complex occupied by b5. These data suggest that a specific allosteric binding site on b5, which includes residues E48, E49, and possibly R52, mediates the stimulation of 17,20-lyase activity.     

Missense mutation serine106----proline causes 17 alpha-hydroxylase deficiency.

Steroid 17 alpha-hydroxylase deficiency is caused by defects in cytochrome P450c17, the single enzyme that has 17-alpha hydroxylase and 17,20-lyase activities. We describe a rapid and efficient polymerase chain reaction tactic for identifying these genetic lesions and identify Ser106----Pro as the cause of 17 alpha-hydroxylase deficiency in two unrelated homozygous patients from Guam. We used site-directed mutagenesis of the normal P450c17 cDNA to construct the Pro106 mutant, and expressed both the normal and mutant sequences in monkey COS-1 cells and in yeast. Expression of the normal sequence permitted the cells to convert pregnenolone to 17-OH pregnenolone, progesterone to 17-OH progesterone, and 17-OH pregnenolone to dehydroepiandrosterone, showing the normal sequence conferred both 17 alpha-hydroxylase and 17,20-lyase activities. Expression of the mutant sequence generated P450c17 mRNA, but conferred none of these activities, proving that the Ser106----Pro mutation abolished the 17 alpha-hydroxylase and 17,20-lyase activities. An HhaI restriction site created by the mutation should permit screening of large populations.     

New compound heterozygous mutation in the CYP17 gene in a 46,XY girl with 17 alpha-hydroxylase/17,20-lyase deficiency

BACKGROUND: 17alpha-Hydroxylase/17,20-lyase deficiency is caused by a defect of P450c17 which catalyzes both 17alpha-hydroxylase and 17,20-lyase reactions in adrenal glands and gonads. RESULTS: In the present study, we analyzed the CYP17 gene in a Japanese patient with 17alpha-hydroxylase/17,20-lyase deficiency. The patient was a phenotypic girl and referred to us for right-sided inguinal hernia at the age of 4 years. Biopsy of the herniated gonad showed testicular tissue. The karyotype was 46,XY. At 6 years of age, hypertension was clearly recognized and the patient was diagnosed as having 17alpha-hydroxylase/17,20-lyase deficiency based on the clinical and laboratory findings. Analysis of the CYP17 gene revealed a compound heterozygous mutation. One mutation was an undescribed single nucleotide deletion at codon 247 in exon 4 (CTT to CT: 247delT) and the other was a missense mutation resulting in a substitution of His to Leu at codon 373 in exon 6 (CAC to CTC: H373L), which has been previously shown to abolish both 17alpha-hydroxylase and 17,20-lyase activities. The functional expression study of the 247delT mutant showed that this 247delT mutation completely eliminates both 17alpha-hydroxylase and 17,20-lyase activities. CONCLUSIONS: Together, these results indicate that the patient is a compound heterozygote for the mutation of the CYP17 gene (247delT and H373L) and that these mutations inactivate both 17alpha-hydroxylase and 17,20-lyase activities and give rise to clinically manifest 17alpha-hydroxylase/17,20-lyase deficiency.     

Homozygous CYP17A1 mutation (H373L) identified in a 46,XX female with combined 17α-hydroxylase/17,20-lyase deficiency

Background: Defects in cytochrome P450c17 are uncommon forms of congenital adrenal hyperplasia caused by CYP17A1 mutations. An H373L mutation in the CYP17A1 gene has been identified in Japanese and Chinese patients. This mutation impairs 17α-hydroxylase and 17,20-lyase activity. Case: A 23-year-old Korean female (46,XX) presented with absent spontaneous puberty and hypertension. Hormonal findings were consistent with combined 17α-hydroxylase/17,20-lyase deficiency. Very high levels of progesterone and 11-deoxycorticosterone were detected, coincident with normal 17-hydroxysteroid levels. Plasma levels of dehydroepiandrosterone, androstenedione and testosterone were extremely low. Mutation analysis of the CYP17A1 gene identified a homozygous missense mutation changing His (CAC) to Leu (CTC) at codon 373. This mutation is known to completely abolish both 17α-hydroxylase and 17,20-lyase activity. The patient's nonconsanguineous parents were heterozygous for this mutation. Of note, her serum steroid levels indicated decreased, but still present, 17α-hydroxylase activity in vivo. Conclusion: We detected a homozygous H373L mutation in a patient with combined 17α-hydroxylase/17,20-lyase deficiency. Our findings demonstrate minimally preserved 17α-hydroxylase activity in vivo and contribute to our knowledge of the regional prevalence of this mutation in Northeast Asia.     

Thiazolidinediones but not metformin directly inhibit the steroidogenic enzymes P450c17 and 3beta -hydroxysteroid dehydrogenase

Androgen biosynthesis requires 3beta-hydroxysteroid dehydrogenase type II (3betaHSDII) and the 17alpha-hydroxylase and 17,20-lyase activities of cytochrome P450c17. Thiazolidinedione and biguanide drugs, which are used to increase insulin sensitivity in type 2 diabetes, lower serum androgen concentrations in women with polycystic ovary syndrome. However, it is unclear whether this is secondary to increased insulin sensitivity or to direct effects on steroidogenesis. To investigate potential actions of these drugs on P450c17 and 3betaHSDII, we used "humanized yeast" that express these steroidogenic enzymes in microsomal environments. The biguanide metformin had no effect on either enzyme, whereas the thiazolidinedione troglitazone inhibited 3betaHSDII (K(I) = 25.4 +/- 5.1 microm) and both activities of P450c17 (K(I) for 17alpha-hydroxylase, 8.4 +/- 0.6 microm; K(I) for 17,20-lyase, 5.3 +/- 0.7 microm). The action of troglitazone on P450c17 was competitive, but it was mainly a noncompetitive inhibitor of 3betaHSDII. The thiazolidinediones rosiglitazone and pioglitazone exerted direct but weaker inhibitory effects on both P450c17 and 3betaHSDII. These differential effects of the thiazolidinediones do not correlate with their effects on insulin sensitivity, suggesting that distinct regions of the thiazolidinedione molecule mediate these two actions. Thus, thiazolidinediones inhibit two key enzymes in human androgen synthesis contributing to their androgen-lowering effects, whereas metformin affects androgen synthesis indirectly, probably by lowering circulating insulin concentrations.     

CYP17 mutation E305G causes isolated 17,20-lyase deficiency by selectively altering substrate binding

Cytochrome p450c17 (CYP17) converts the C21 steroids pregnenolone and progesterone to the C19 androgen precursors dehydroepiandrosterone (DHEA) and androstenedione, respectively, via sequential 17alpha-hydroxylase and 17,20-lyase reactions. Disabling mutations in CYP17 cause combined 17alpha-hydroxylase/17,20-lyase deficiency, but rare missense mutations cause isolated loss of 17,20-lyase activity by disrupting interactions of redox partner proteins with CYP17. We studied an adolescent male with clinical and biochemical features of isolated 17,20-lyase deficiency, including micropenis, hypospadias, and gynecomastia, who is homozygous for CYP17 mutation E305G, which lies in the active site. When expressed in HEK-293 cells or Saccharomyces cerevisiae, mutation E305G retains 17alpha-hydroxylase activities, converting pregnenolone and progesterone to 17alpha-hydroxysteroids. However, mutation E305G lacks 17,20-lyase activity for the conversion of 17alpha-hydroxypregnenolone to DHEA, which is the dominant pathway to C19 steroids catalyzed by human CYP17 (the delta5-steroid pathway). In contrast, mutation E305G exhibits 11-fold greater catalytic efficiency (kcat/Km) for the cleavage of 17alpha-hydroxyprogesterone to androstenedione compared with wild-type CYP17. We conclude that mutation E305G selectively impairs 17,20-lyase activity for DHEA synthesis despite an increased capacity to form androstenedione. Mutation E305G provides genetic evidence that androstenedione formation from 17alpha-hydroxyprogesterone via the minor delta4-steroid pathway alone is not sufficient for complete formation of the male phenotype in humans.     

17α-Hydroxylase/17,20-lyase defects

Congential adrenal hyperplasia due to 17α-hydroxylase/17/20-lyase deficiency is caused by genetic defects in the gene encoding P450c17 (CYP17). To date, 18 different mutations in 27 individuals have been identified and all of them are located in the coding region of CYP17. Several mutations have been reconstructed in human P450c17 cDNA and expressed in COS 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 result from complete or partial combined deficiencies of 17α-hydroxylase/17,20-lyase. The elucidation of the molecular bases generally explains the patient's clinical profiles including the sexual phenotype of the external genitalia. In one case initially 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. However, the patient was subsequently found to have 17α-hydroxylase deficiency, suggesting involvements of age-dependent unknown factors affecting P450c17 activity.     

Novel mutation of the CYP17 gene in two unrelated patients with combined 17alpha-hydroxylase/17,20-lyase deficiency: demonstration of absent enzyme activity by expressing the mutant CYP17 gene and by three-dimensional modeling

The CYP17 gene, located on chromosome 10q24-q25, encodes the cytochrome P450c17 enzyme. Mutations of this gene cause the 17alpha-hydroxylase/17,20-lyase deficiency, which is a rare, autosomal recessive form of congenital adrenal hyperplasia. Approximately 50 different mutations of the CYP17 gene have been described, of which some mutations have been identified in certain ethnic groups. In this study, we present the clinical history, hormonal findings and mutational analysis of two patients from unrelated families, who were evaluated for hypertension, hypokalemia and sexual infantilism. In the first patient, who was a 37-year-old female, additional studies showed a large myelolipoma in the left adrenal gland, and a smaller tumor in the right adrenal gland. In the second patient, who was a 31-year-old phenotypic female, clinical work-up revealed a 46,XY kariotype, absence of ovaries and presence of testes located in the inner opening of both inguinal canals. Analysis of the CYP17 gene by polymerase chain reaction amplification and direct sequencing demonstrated a novel homozygous mutation of codon 440 from CGC (Arg) to TGC (Cys) in both patients. The effect of this novel mutation on 17alpha-hydroxylase/17,20-lyase activity was assessed by in vitro studies on the mutant and wild-type P450c17 generated by site-directed mutagenesis and transfected in nonsteroidogenic COS-1 cells. These studies showed that the mutant P450c17 protein was produced in transfected COS-1 cells, but it had negligible 17alpha-hydroxylase and 17,20-lyase activities. In addition, three-dimensional computerized modeling of the heme-binding site of the P450c17 enzyme indicated that replacement of Arg by Cys at amino acid position 440 predicts a loss of the catalytic activity of the enzyme, as the mutant enzyme containing Cys440 fails to form a hydrogen bond with the propionate group of heme, which renders the mutant enzyme unable to stabilize the proper position of heme. Based on these findings we conclude that expressing the CYP17 gene with functional analysis, combined with three-dimensional computerized modeling of the heme-binding site of the protein provide feasible tools for molecular characterizing of functional consequences of the novel CYP17 mutation on enzyme function.     

Deletion of P399_E401 in NADPH cytochrome P450 oxidoreductase results in partial mixed oxidase deficiency

P450 oxidoreductase (POR) is the electron donor for all microsomal P450s including steroidogenic enzymes CYP17A1, CYP19A1 and CYP21A2. We found a novel POR mutation P399_E401del in two unrelated Turkish patients with 46,XX disorder of sexual development. Recombinant POR proteins were produced in yeast and tested for their ability to support steroid metabolizing P450 activities. In comparison to wild-type POR, the P399_E401del protein was found to decrease catalytic efficiency of 21-hydroxylation of progesterone by 68%, 17α-hydroxylation of progesterone by 76%, 17,20-lyase action on 17OH-pregnenolone by 69%, aromatization of androstenedione by 85% and cytochrome c reduction activity by 80%. Protein structure analysis of the three amino acid deletion P399_E401 revealed reduced stability and flexibility of the mutant. In conclusion, P399_E401del is a novel mutation in POR that provides valuable genotype–phenotype and structure–function correlation for mutations in a different region of POR compared to previous studies. Characterization of P399_E401del provides further insight into specificity of different P450s for interaction with POR as well as nature of metabolic disruptions caused by more pronounced effect on specific P450s like CYP17A1 and aromatase.     

The enantiomer of progesterone (ent-progesterone) is a competitive inhibitor of human cytochromes P450c17 and P450c21

Human cytochrome P450c17 (17alpha-hydroxylase, 17,20-lyase) (CYP17) and cytochrome P450c21 (21-hydroxylase) (CYP21) differ by only 14 amino acids in length and share 29% amino acid identity. Both enzymes hydroxylate progesterone at carbon atoms that lie only 2.6A apart, but CYP17 also metabolizes other steroids and demonstrates additional catalytic activities. To probe the active site topologies of these related enzymes, we synthesized the enantiomer of progesterone and determined if ent-progesterone is a substrate or inhibitor of CYP17 and CYP21. Neither enzyme metabolizes ent-progesterone; however, ent-progesterone is a potent competitive inhibitor of CYP17 (K(I)=0.2 microM). The ent-progesterone forms a type I difference spectrum with CYP17, but molecular dynamics simulations suggest different binding orientations for progesterone and its enantiomer. The ent-progesterone also inhibits CYP21, with weaker affinity than for CYP17. We conclude that CYP17 accommodates the stereochemically unnatural ent-progesterone better than CYP21. Enantiomeric steroids can be used to probe steroid binding sites, and these compounds may be effective inhibitors of steroid biosynthesis.     

Assessment of the ability of type 2 cytochrome b5 to modulate 17,20-lyase activity of human P450c17

The 17 alpha-hydroxylase and 17,20-lyase activities of P450c17 lead to the production of 17 alpha-hydroxypregnenolone (17 alpha-OH-Preg) and dehydroepiandrosterone (DHEA), respectively, in different tissues. The mechanisms of differential regulation of these two activities are not yet fully elucidated. It has been previously shown that cytochrome b5 (cyt-b5) could facilitate the 17,20-lyase activity of human P450c17. Recently, a cDNA (type 2 cyt-b5) sharing 45.8% homology with type 1 cyt-b5 has been isolated from human testis. Since high 17,20-lyase activity is required for the production of androgens in the testis, we wanted to determine the importance of this second cDNA in the modulation of P450c17 17,20-lyase activity and hence, its role in the formation of active androgens. We therefore isolated type 2 cyt-b5 from human testis by RT-PCR and analyzed, by transient transfection in transformed human embryonic kidney cells (HEK-293) of various amounts of vectors expressing cyt-b5, P450-reductase and P450c17, its ability to modulate the 17,20-lyase activity of human P450c17. Results show that, in the presence of NADPH cytochrome P450 reductase (P450-red), type 2 cyt-b5 increases 17,20-lyase activity to a level comparable to that of type 1. These results support the idea that types 1 and 2 cyt-b5 could be involved in the differential modulation of 17 alpha-hydroxylase and 17,20-lyase activities of P450c17. Furthermore, the analysis of mRNA expression of types 1 and 2 cyt-b5 by RT-PCR using primers specific to each type showed that both types are present in the liver but also in the adrenal and testis.     

Molecular basis of apparent isolated 17,20-lyase deficiency: compound heterozygous mutations in the C-terminal region (Arg(496)----Cys, Gln(461)----Stop) actually cause combined 17 alpha-hydroxylase/17,20-lyase deficiency.

The molecular defect in a reported case of isolated 17,20-lyase deficiency in a 46XY individual has been elucidated. The patient was found to be a compound heterozygote, carrying two different mutant alleles in the CYP17 gene. One allele contains a point mutation of arginine (CGC) to cysteine (TGC) at amino acid 496 in exon 8. The second allele contains a stop codon (TAG) in place of glutamine (CAG) at position 461 in exon 8 which is located 19 amino acids to the carboxy-terminal side of the P-450(17) alpha heme binding cysteine. COS-1 cells transfected with cDNAs containing one or the other of these mutations showed dramatically reduced 17 alpha-hydroxylase and 17,20-lyase activities relative to cells transfected with the wild type P-450(17) alpha cDNA. While the in vitro data in COS 1 cells can explain the patient's physical phenotype, with female external genitalia, it was somewhat discordant with the clinical expression of isolated 17,20-lyase deficiency with relative preservation of 17 alpha-hydroxylase activity in vivo. In addition to the expression studies of these two examples of mutants in the C-terminal region of cytochrome P-450(17) alpha, a third mutant cDNA construct containing a 4-base duplication at codon 480 previously found in patients with combined 17 alpha-hydroxylase/17,20-lyase deficiency was also expressed in COS-1 cells. This expressed protein was completely inactive with respect to both activities, supporting the biochemical findings in serum and in vitro biochemical data obtained using a testis from the patient. The results from these patients clearly indicate the importance of the C-terminal region of human P-450(17) alpha in its enzymatic activities.     

Assessment of porcine and human 16-ene-synthase, a third activity of P450c17, in the formation of an androstenol precursor. Role of recombinant cytochrome b5 and P450 reductase.

Recently, we have shown that the biosynthesis of androstenol, a potential endogenous ligand for the orphan receptors constitutive androstane receptor and pregnane-X-receptor, requires the presence of enzymes of the steroidogenic pathway, such as 3 beta-hydroxysteroid dehydrogenase, 5 alpha-reductase and 3 alpha-hydroxysteroid dehydrogenase. In this report, we examine at the molecular level whether the enzyme 17 alpha-hydroxylase/17,20-lyase (P450c17), which possesses dual 17 alpha-hydroxylase and 17,20-lyase activities and catalyzes the production of precursors for glucocorticoids and sex steroids, is also able to catalyze the formation of a third class of active steroids, 16-ene steroids (including androstenol). The role of components of the P450 complex is also assessed. We transfected human embryonic kidney (HEK-293) cells with various amounts of vectors expressing P450c17, NADPH-cytochrome P450 reductase, and cytochrome b5. Our results showed that P450c17 possesses a 16-ene-synthase activity able to transform pregnenolone into 5,16-androstadien-3 beta-ol, without the formation of the precursor 17-hydroxypregnenolone. Cytochrome b5 has a much stronger effect on the 16-ene-synthase activity than on the 17 alpha-hydroxylase/17,20-lyase activities. On the other hand, P450reductase has a drastic effect on the latter, but a negligible one on 5,16-androstadien-3 beta-ol synthesis. Our results therefore demonstrate that human P450c17, as other enzymes of the classical steroidogenic pathway, is involved in the biosynthetic pathway leading to the formation of androstenol.     

Morphologic change and elevation of cortisol secretion in cultured human normal adrenocortical cells caused by mutant p21K-ras protein

In our previous study on the tumorigenesis of human functional adrenal tumors, we observed a high frequency of K-ras point mutations in clinical specimens. Furthermore, we cloned the mutated K-ras gene from the tumors and inserted it into vectors to transfect normal bovine adrenocortical cells to express the mutated K-ras gene. The mRNA level of steroidogenic enzymes such as cholesterol sidechain cleavage enzyme (P450SCC), 17alpha-hydroxylase/17,20-lyase (P450c17), and 3beta-hydroxysteroid dehydrogenase (3betaHSD) in the mutant K-ras stably transfected cells were elevated. Cultured normal adrenocortical cells from donors and patients with adrenocortical tumors were then transfected with mutant K-ras expression plasmids constructed from human adrenal tumors. Stable transfectants grew faster than normal cells. Additionally, morphologic change was observed in the transfected cells. Moreover, when the synthesis of hormones was analyzed, the mRNA of P450SCC, P450C17, and 3betaHSD was found to have increased, and the level of cortisol was 18 to 25 times that in control cells. The increased steroid hormone production in mutant K-ras-transfected cells was reversed by lovastatin, a pharmacologic inhibitor of p21ras function. These results, combined with previous reports of steroidogenic K-ras in bovine adrenocortical cells, suggest that the K-ras oncogene is involved in steroidogenesis in human adrenocortical cells.     

Differential inhibition of CYP17A1 and CYP21A2 activities by the P450 oxidoreductase mutant A287P

P450 oxidoreductase (POR) has a pivotal role in facilitating electron transfer from nicotinamide adenine dinucleotide phosphate to microsomal cytochrome P450 (CYP) enzymes, including the steroidogenic enzymes CYP17A1 and CYP21A2. Mutations in POR have been shown recently to cause congenital adrenal hyperplasia with apparent combined CYP17A1 and CYP21A2 deficiency that comprises a variable clinical phenotype, including glucocorticoid deficiency, ambiguous genitalia, and craniofacial malformations. To dissect structure-function relationships potentially explaining this phenotypic diversity, we investigated whether specific POR mutations have differential effects on CYP17A1 and CYP21A2. We compared the impact of missense mutations encoding for single amino acid changes in three distinct regions of the POR molecule: 1), Y181D and H628P close to the central electron transfer area, 2) S244C located within the hinge close to the flavin adenine dinucleotide and flavin mononucleotide domains of POR, and 3) A287P that is clearly distant from the two other regions. Functional analysis using a yeast microsomal assay with coexpression of human CYP17A1 or CYP21A2 with wild-type or mutant human POR revealed equivalent decreases in CYP17A1 and CYP21A2 activities by Y181D, H628P, and S244C. In contrast, A287P had a differential inhibitory effect, with decreased catalytic efficiency (Vmax/Km) for CYP17A1, whereas CYP21A2 retained near normal activity. In vivo analysis of urinary steroid excretion by gas chromatography/mass spectrometry in 11 patients with POR mutations showed that A287P homozygous patients had the highest corticosterone/cortisol metabolite ratios, further indicative of preferential inhibition of CYP17A1. These findings provide novel mechanistic insights into the redox regulation of human steroidogenesis. Differential interaction of POR with electron-accepting CYP enzymes may explain the phenotypic variability in POR deficiency, with additional implications for hepatic drug metabolism by POR-dependant CYP enzymes.     

Recent aspects of steroid biosynthesis in male sex differentiation. Clinical studies.

Recent discoveries in molecular biology have much clarified the regulation and function of steroid-converting enzymes. Most progress has been made in the area of cytochromes, which regulate the side chain cleavage of cholesterol (P-450 SCC) and the 17 alpha-hydroxylase and 17,20-desmolase (or 17,20-lyase) activities (P-450 17 alpha), as well as in 3 beta-hydroxysteroid dehydrogenase. Nevertheless, there are some discrepancies between fundamental knowledge and clinical experience, which are difficult to understand: why is it for example possible that cases with 'pure' 17 alpha-hydroxylase or 17,20-desmolase deficiency exist, when there is only one cytochrome regulating both steps? After a brief review of clinical and biochemical findings in the various defects of testosterone biosynthesis, a case is discussed, which is of interest in this respect. This XY patient with female external genitalia, who has been shown to have compound heterozygous mutations, had 'pure' 17,20-desmolase deficiency up to adolescence, but additional 17 alpha-hydroxylase deficiency with hypertension developed thereafter. From this observation, it has to be concluded that as yet unknown, possibly age-dependent modulating factors exist, which influence the activity of the cytochrome. Also the estrogen replacement given to the patient might have played a role in this change.