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.     

Deletion of a phenylalanine in the N-terminal region of human cytochrome P-450(17 alpha) results in partial combined 17 alpha-hydroxylase/17,20-lyase deficiency

Steroid 17 alpha-hydroxylase and 17,20-lyase activities reside within the same polypeptide chain (cytochrome P-450(17 alpha)), and consequently human 17 alpha-hydroxylase deficiencies are characterized by defects in either or both of these activities. Human mutants having these deficiencies represent an excellent source of material for investigation of P-450(17 alpha) structure-function relationships. The CYP17 gene from an individual having partial combined 17 alpha-hydroxylase/17,20-lyase deficiency has been characterized structurally and the homozygous mutation found to be the deletion of the phenylalanine codon (TTC) at either amino acid position 53 or 54 in exon 1. Reconstruction of this mutation into a human P-450(17 alpha) cDNA followed by expression in COS 1 cells led to production of the same amount of immunodetectable P-450(17 alpha) protein as found with expression of the normal human P-450(17 alpha) cDNA. However, 17 alpha-hydroxylase activity of this mutant protein measured in intact cells was less than 37% of that observed upon expression of the wild-type enzyme, whereas 17,20-lyase activity of the mutant was less than 8% of that observed with the normal enzyme. When estimated in intact cells, the Km for 17 alpha-hydroxylation of progesterone was increased by a factor of 2 in the mutant enzyme, whereas the Vmax was reduced by a factor of 3. In order to estimate the kinetic parameters for the 17,20-lyase reaction, microsomes were isolated from transfected COS 1 cells to enrich for this activity. Surprisingly, the specific activity of the mutant 17 alpha-hydroxylase in microsomes was 3-fold less than that observed in intact cells, indicating that the structure of mutant P-450(17 alpha) was dramatically altered upon disruption of COS 1 cells. Apparently the deletion of a single phenylalanine in the N-terminal region of P-450(17 alpha) alters its folding in such a way that both enzymatic activities are dramatically decreased, leading to the partial combined deficiency observed in this individual.     

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.     

C21 steroid side chain cleavage enzyme from porcine adrenal microsomes. Purification and characterization of the 17 alpha-hydroxylase/C17,20-lyase cytochrome P-450

The properties and the purity of a cytochrome P-450 (17 alpha-hydroxylase) from porcine adrenal microsomes have been examined following a report that the corresponding enzyme from bovine adrenocortical microsomes is inactive as a 17 alpha-hydroxylase and fails to show a high spin spectrum on addition of substrate, once the enzyme has been purified (Bumpus, J. A., and Dus, K. M. (1982) J. Biol. Chem. 257, 12696-12704). The purity of the porcine enzyme was demonstrated by electrophoresis on polyacrylamide with sodium dodecyl sulfate, immunoelectrophoresis, and NH2-terminal amino acid sequence (16 residues). The pure enzyme shows Mr = 54,000, heme content of greater than 0.8 nmol/nmol of protein, and absorption spectra typical of cytochrome P-450. The enzyme is active with both delta 4 (progesterone) and delta 5 (pregnenolone) substrates as a 17 alpha-hydroxylase and with the corresponding 17 alpha-hydroxysteroids as a C17,20-lyase. All four substrates produce typical type I spectra with the enzyme (so-called high spin form). We conclude that: 1) porcine adrenal microsomes contain a 17 alpha-hydroxylase/C17,20-lyase which is a single protein molecule readily purified to an enzymatically active form; 2) the C17,20-lyase activity is largely suppressed in the microsomes; and 3) the enzyme closely resembles that found in testicular microsomes. We propose that this enzyme be referred to as the adrenal C21 steroid side chain cleavage enzyme.     

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.     

Defects in steroidogenic enzymes. Discrepancies between clinical steroid research and molecular biology results

Molecular biology has clarified the understanding of steroidogenic enzyme genetics. Nevertheless, there are discrepancies between fundamental and clinical experience. (1) Why do patients with “pure” 17-hydroxylase or 17,20-desmolase deficiency exist, when one cytochrome regulates both steps? A case of interest is discussed, who had “pure” 17,20-desmolase deficiency until adolescence, but additional 17-hydroxylase deficiency thereafter. (2) In 11β-hydroxylase deficiency, it was puzzling to find 18-hydroxylated compounds, and, in isolated hypoaldosteronism, normal cortisol, since 11β- and 18-hydroxylation were thought to be regulated together. This has now been explained by differences in the fasciculata and glomerulosa. The occurrence of 11β-hydroxylase deficiency of 17-hydroxylated steroids only, however, remains enigmatic. (3) 3β-Hydroxysteroid dehydrogenase deficiency does not only seem to exist in classic (mutations of type II gene), but also in late-onset cases. In them, no molecular basis could be found. (4) Also, in cholesterol side-chain cleavage, there is an inequity: while evidently one cytochrome regulates 20- and 22-hydroxylation, pregnenolone is formed when 20OH-cholesterol, but not when cholesterol, is added to adrenal tissue of deficient patients. Other factors (promoters, fusion proteins, adrenodoxin, cAMP-dependent expression of genes, and/or proteases), or hormonal replacement in patients may be responsible for these discrepancies.     

Immunochemical studies on cytochrome P-450 in adrenal microsomes

An antibody was prepared against electrophoretically homogeneous cytochrome P-450C21 purified from bovine adrenal microsomes. This antibody was used to compare various cytochromes P-450 in bovine and guinea pig adrenal microsomes. In an Ouchterlony double diffusion test, a spur formation was observed between the precipitin lines of the purified bovine cytochrome P-450C21 and guinea pig adrenal microsomes against anti-cytochrome P-450C21 IgG. Anti-cytochrome P-450C21 IgG inhibited 21-hydroxylation both of bovine and guinea pig adrenal microsomes but the inhibition was much more effective in the bovine microsomes than in the guinea pig microsomes. These results suggest that the 21-hydroxylase in the guinea pig microsomes has some molecular similarities to the bovine cytochrome P-450C21 and a part of the antibodies cross-reacts with the 21-hydroxylase in the guinea pig microsomes. Anti-cytochrome P-450C21 IgG did not inhibit the activities of 17 alpha-hydroxylase and C17,20-lyase in the bovine and guinea pig microsomes but stimulated these activities. This result shows that different species of cytochrome P-450 other than cytochrome P-450C21 catalyzes the 17 alpha-hydroxylation and C17,20 bond cleavage. The stimulation of 17 alpha-hydroxylation and C17,20 bond cleavage by blocking 21-hydroxylation indicates that the electron transfer systems for various cytochromes P-450 are intimately linked in adrenal microsomes.     

Nucleotide sequence of a cDNA encoding porcine testis 17 alpha-hydroxylase cytochrome P-450.

We describe the isolation and characterization of a cDNA encoding the complete porcine neonatal testis 17 alpha-hydroxylase/C-17,20-lyase cytochrome P-450. The deduced amino acid sequence is 509 amino acids in length.     

The regulation of ovine placental steroid 17 alpha-hydroxylase and aromatase by glucocorticoid

Parturition in the pregnant sheep is preceded by an abrupt alteration in placental steroid metabolism causing a shift from progesterone to estrogen production. This change is believed to be a consequence of the prepartum rise in cortisol in the fetal circulation and involves increases in activities of the enzymes steroid 17 alpha-hydroxylase (cytochrome P-450(17)alpha), steroid C-17,20-lyase, and possibly aromatase. We have investigated the activity levels of aromatase and 17 alpha-hydroxylase in placental microsomes in late pregnancy and dexamethasone-induced labor. Over the gestational period of 118-140 days basal levels of placental aromatase were relatively constant [mean value (+/- SD) of 5.6 +/- 1.6 pmol min-1 mg microsomal protein-1 (n = 10)]. Steroid 17 alpha-hydroxylase activity was undetectable [less than 0.5 pmol min-1 mg microsomal protein-1 (n = 7)]. In six animals in labor induced with infusion of dexamethasone into the fetus, placental aromatase activity had a mean value of 14.0 +/- 2.5 pmol min-1 mg protein-1; placental steroid 17 alpha-hydroxylase, measured in four of the animals, had a mean (+/- SD) activity of 319 +/- 58 pmol min-1 mg microsomal protein-1. Immunoblotting of placental microsomal preparations with specific antibodies to cytochrome P-450(17)alpha and NADPH-cytochrome P-450-reductase indicated that the glucocorticoid-induced activity of 17 alpha-hydroxylase was associated with increased content of cytochrome P-450(17)alpha. Northern blotting with a cDNA probe for cytochrome P-450(17)alpha showed that glucocorticoid increased the levels of mRNA for the enzyme.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and biochemical evaluation of a range of potent benzyl imidazole-based compounds as potential inhibitors of the enzyme complex 17alpha-hydroxylase/17,20-lyase (P450(17alpha))

The cytochrome P-450 enzyme, 17alpha-hydroxylase/17,20-lyase (P450(17alpha)), is a potential target in hormone-dependent cancers. Here, we report the synthesis and biochemical evaluation of a range of benzyl imidazole-based compounds which have been targeted against the two components of this enzyme, that is, 17alpha-hydroxylase (17alpha-OHase) and 17,20-lyase (lyase). The results from the biochemical testing suggest that the compounds synthesised are good inhibitors, with N-4-iodobenzyl imidazole (5) (IC50=10.06 microM against 17alpha-OHase and IC50=1.58 microM against lyase) showing equipotent activity against lyase compared to the standard compound, ketoconazole (KTZ) (IC50=3.76+/-0.01 microM against 17alpha-OHase and IC50=1.66+/-0.15 microM against lyase). Furthermore, the compounds tested are less potent towards the 17alpha-OHase component, a desirable property in the development of novel inhibitors of P450(17alpha).     

A novel molecular modelling study of inhibitors of the 17alpha-hydroxylase component of the enzyme system 17alpha-hydroxylase/17,20-lyase (P-450(17alpha)).

The enzyme 17alpha-hydroxylase/17,20-lyase (P-450(17alpha) has recently become the focus of research into the fight against hormone dependent prostate cancer. However, the specific nature of this enzyme, in particular, the dual role of its active site, remains unknown. In our drive to elucidate further information regarding P-450(17alpha), and in light of our experience of other cytochrome P-450 enzymes, we chose to consider each part of this complex enzyme separately (i.e. the 17alpha-hydroxylase (17alpha-OHase) and the 17,20-lyase components). We therefore initiated a series of molecular modelling studies involving the construction of a 'substrate heme complex' for each of the two components. Here, we consider the construction and use of the complex for the 17alpha-OHase component of this enzyme. Using this approach, we have successfully considered: the binding of steroidal and non-steroidal reversible inhibitors: the structural features necessary for potent inhibition: and, rationalised the mode of action of a number of compounds whose inhibitory activity has not been previously explained, for example aminoglutethimide (an inhibitor of another related cytochrome P-450 enzyme, aromatase AR). The study concludes that the ability of the inhibitors of 17alpha-OHase to undergo polar polar interaction with the active site and for the compounds to closely mimic the substrate plane is a major factor in determining potency. Factors such as log P (log of the partition coefficient value for the distribution of a compound between octanol and water) would then appear to determine the extent of overall inhibitory activity. Overall, the study suggests that the novel substrate-heme complex approach has provided a good approximation of the 17alpha-OHase active site and has proved to be a useful tool in drug design and discovery.     

Regulation of 17,20 lyase activity by cytochrome b5 and by serine phosphorylation of P450c17

Cytochrome P450c17 catalyzes the 17alpha-hydroxylase activity required for glucocorticoid synthesis and the 17,20 lyase activity required for sex steroid synthesis. Most P450 enzymes have fixed ratios of their various activities, but the ratio of these two activities of P450c17 is regulated post-translationally. We have shown that serine phosphorylation of P450c17 and the allosteric action of cytochrome b5 increase 17,20 lyase activity, but it has not been apparent whether these two post-translational mechanisms interact. Using purified enzyme systems, we now show that the actions of cytochrome b5 are independent of the state of P450c17 phosphorylation. Suppressing cytochrome b5 expression in human adrenal NCI-H295A cells by >85% with RNA interference had no effect on 17alpha-hydroxylase activity but reduced 17,20 lyase activity by 30%. Increasing P450c17 phosphorylation could compensate for this reduced activity. When expressed in bacteria, human P450c17 required either cytochrome b5 or phosphorylation for 17,20 lyase activity. The combination of cytochrome b5 and phosphorylation was not additive. Cytochrome b5 and phosphorylation enhance 17,20 lyase activity independently of each other, probably by increasing the interaction between P450c17 and NADPH-cytochrome P450 oxidoreductase.     

Steroid interactions with cytochrome P-450 from testis microsomes

The cytochrome P-450 of gonadal microsomes is an integral component of the steroid converting enzymes, 17 alpha-hydroxylase and 17,20-lyase. Interaction of the steroid substrates with this cytochrome results in a shift in the Soret band as measured by difference spectroscopy. In these studies it is shown that in contrast to placental microsomal cytochrome P-450 which binds C19 steroids, testis microsomal cytochrome P-450 primarily binds C21 steroids. However, addition of a 17 alpha- methyl, 17 beta-acetate or a 17 beta-benzoate group to testosterone permits interaction. The addition of hydroxyl or methyl groups to other positions does not affect binding. The presence of multiple oxygen functions on C21 steroids, as in cortisol and corticosterone, precludes interaction. At least one oxygen function seems necessary for binding as 5 alpha- and 5 beta-pregnane do not bind whereas 20-deoxypregnenolone (5-pregnen-3 beta-ol) does bind. These findings indicate that factors in addition to hydrophobic interactions dictate the binding of steroid substrates to testis microsomal cytochrome P-450.     

Characterization of complementary deoxyribonucleic acid for human adrenocortical 17 alpha-hydroxylase: a probe for analysis of 17 alpha-hydroxylase deficiency

To provide a basis for investigation of the molecular mechanisms underlying the hormonal regulation of steroid 17 alpha-hydroxylase (P-450 17 alpha) activity in adrenal, ovary, and testis as well as human 17 alpha-hydroxylase deficiency, we have isolated from a human fetal adrenal cDNA library a cDNA sequence complementary to the mRNA that encodes the human P-450 17 alpha enzyme. Of 75,000 colonies from the library that were screened by use of a nick-translated 5'-specific bovine P-450 17 alpha cDNA probe, 10 positive colonies were isolated and the clone with the longest insert (pcD-17 alpha H) was selected for further characterization. pcD-17 alpha H encodes the complete human P-450 17 alpha protein having approximately 78% homology at the nucleotide level and 71% homology at the amino acid level when the sequence of pcD-17 alpha H is compared to the bovine P-450 17 alpha cDNA sequence. By transient expression of the human P-450 17 alpha cDNA clone in COS 1 cells, we have demonstrated that the 17 alpha-hydroxylase and 17,20 lyase activities reside within the same human P-450 17 alpha polypeptide chain. The insert was also used as a probe to investigate, by means of Southern blot analysis, possible alterations in the P-450 17 alpha gene sequence in DNA isolated from skin fibroblasts from three patients with clinically characterized 17 alpha-hydroxylase deficiencies. No changes were detected in the DNA of any of the patients by this analysis.     

Heterogeneity of cytochrome P-450 in rat testis microsomes.

Cytochrome P-450 appears to be a component of the steroid-coverting enzymes, 17alpha-hydroxylase and 17,20-lyase, which catalyze sequential steps in sex hormone synthesis. Further evidence indicates that the steroid substrates of these enzymes bind to cytochrome P-450 during catalysis. The present report deals with the problem of whether a single form of cytochrome P-450 mediates both enzyme reactions or whether two enzymes are involved. Both activities are competitively inhibited by a number of the same inhibitors. Because K1 values of competitive inhibitors are dissociated constants, and thus a property of the cytochrome, different magnitudes of K1, determined for the same inhibitor with each enzyme, are consistent with the participation of more than one form of cytochrome P-450. Differences in the K1 values were found to be statistically significant and varied from 3- to 10-fold. Two competitive inhibitors retarded velocities with one reaction but not the other. In addition, the enzyme activities were markedly different in their sensitivity to carbon monoxide inhibition. The conclusion based on these two lines of evidence is that separate enzymes and different forms of cytochrome P-450 are involved in each reaction.     

Destruction of testicular cytochrome P-450 by 7 alpha-thiospironolactone is catalyzed by the 17 alpha-hydroxylase.

Studies were done to determine the role of the 17 alpha-hydroxylase in the conversion of 7 alpha-thiospironolactone (7 alpha-thio-SL) to a reactive metabolite causing the degradation of testicular cytochrome P-450. Incubation of guinea pig testicular microsomes with 7 alpha-thio-SL plus NADPH resulted in an approx. 70% decline in cytochrome P-450 content and even greater loss of 17 alpha-hydroxylase activity. Addition of the 17 alpha-hydroxylase inhibitor, SU-10'603, to the incubation medium prevented the degradation of P-450 by 7 alpha-thio-SL. Similarly, preincubation of testicular microsomes with anti-P-45017 alpha,lyase IgG to inhibit 17 alpha-hydroxylation, diminished the subsequent loss of P-450 caused by 7 alpha-thio-SL. The results indicate that the 17 alpha-hydroxylase catalyzes the conversion of 7 alpha-thio-SL to the reactive metabolite responsible for P-450 destruction. The accompanying loss of 17 alpha-hydroxylase activity supports the hypothesis that suicide inhibition is the mechanism involved.     

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.     

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.