Purification and characterization of bovine steroid 21-hydroxylase (P450c21) efficiently expressed in Escherichia coli

Steroid 21-hydroxylase, P450c21, is responsible for the conversion of progesterone and 17alpha-hydroxyprogesterone to their 21-hydroxylated derivatives. P450c21 has been poorly investigated because of difficulty in obtaining sufficient quantities of purified protein. To solve the problem, we have attempted to express the bovine P450c21 in Escherichia coli as a stable form. The N-terminal membrane anchor and basic regions of P450c21 were replaced by the basic region of CYP2C3. The engineered P450c21 was expressed at a level higher than 1.2micromol/L culture (>60mg/L) when coexpressed with molecular chaperones GroES/GroEL. Utilizing three steps of column chromatography, the protein was highly purified to the specific content 16.6nmol/mg (91.2% purity). The purified protein is a monomer in the presence of 1% sodium cholate as determined by gel filtration analysis, suggesting that this membrane anchor-truncated form of P450c21 is more soluble than the native form. The purified enzyme showed typical substrate-binding difference spectra and 21-hydroxylase activities for both progesterone and 17alpha-hydroxyprogesterone. Truncation of the membrane anchor increases solubility of P450c21 facilitating expression of this protein in E. coli yielding sufficient quantities for both biochemical and biophysical studies.     

Phytoestrogen resveratrol suppresses steroidogenesis by rat adrenocortical cells by inhibiting cytochrome P450 c21-hydroxylase

BACKGROUND AND AIM: The phytoestrogen resveratrol is found in grapes, mulberries and peanuts, all of which are consumed regularly by humans. Resveratrol is also used in chemotherapy against cancer and aging and as a cardioprotectant. The aim of the present study was to characterize the effects of resveratrol on rat adrenal steroidogenesis and to study the underlying mechanism. METHODS: Adrenocortical cells were isolated from the adrenal glands of normal male rats (in vitro) and from male rats administered resveratrol in their diet for 12 weeks (ex vivo). Cells from resveratrol-treated and non-treated rats were tested ex vivo for responsiveness to ACTH and cells from normal rats were tested in vitro for responsiveness to ACTH in the presence and absence of resveratrol. Corticosterone and progesterone production were measured by RIA and expression of steroidogenic enzymes analyzed by PAGE/Western blotting. RESULTS: Corticosterone production was inhibited 47% by 50 microM resveratrol in vitro and 20% ex vivo, while progesterone production was elevated to 400% of the control value in in vitro experiments. Resveratrol treatment decreased adrenal cytochrome P450 c21-hydroxylase expression in vivo and cell culture conditions. No changes in cell viability or morphology were caused by exposure to resveratrol in both ex vivo and in vitro experiments. CONCLUSION: Resveratrol suppresses corticosterone production by primary rat adrenocortical cell cultures in vitro and ex vivo by inhibiting cytochrome P450 c21-hydroxylase.     

Evidence for frequent gene conversion in the steroid 21-hydroxylase P-450(C21) gene: implications for steroid 21-hydroxylase deficiency.

Oligonucleotide probes specific for the deleterious mutations harbored in the P-450(C21)A pseudogene and oligonucleotide probes specific for the corresponding sequences in the B gene were prepared to examine the molecular lesions in the P-450(C21) gene of P-450(C21)-deficient patients. Using these gene-specific probes, we performed Southern blot analyses of genomic DNAs from 11 patients and eight normal individuals. At least one allele of the B gene (the 3.7-kb TaqI fragment) in a patient was inactivated by mutations caused by recombination with the A gene. The A genes in normal individuals and patients seemed to be replaced frequently (i.e., 10/19 individuals) in their 3' portions by B gene sequences. All of these alterations occurred without changing the characteristic length (3.2 kb) of the TaqI fragment of the A gene, a result strongly suggesting that frequent gene conversions and/or intragenic recombinations have happened in the P-450(C21) genes. Densitometric analysis of the autoradiograms from hybridization experiments revealed extensive variation (from one to five copies) in the copy number of the A gene (the 3.2-kb TaqI fragment) whereas that of the B gene (the 3.7-kb TaqI fragment) was relatively constant at two or three copies.     

Analysis of steroid 21-hydroxylase gene in five unrelated Japanese patients with 21-hydroxylase deficiency

DNA samples from five unrelated Japanese patients with 21-hydroxylase (21-OHase) deficiency were studied by Southern analysis using human 21-OHase cDNA. Patterns seen after digestion with not only TaqI but also KpnI showed that two out of the five patients were homozygous for a deletion of the 21-OHase B gene. This result supports the report that the 21-OHase B gene is functional. In the other three, smaller mutations might be responsible for the disorder. The parents of one of the two patients with the deletion had a common ancestor. Hybridization patterns of DNA from members of the family of the patient were consistent with an autosomal recessive mode of inheritance of the deletion that correlates with the clinical phenotype. The deletion segregated with HLA-Aw 24; Bw 61; Cw 3. Heterozygous carriers of 21-OHase deficiency could be detected by comparing the patterns as well as the HLA haplotypes in this family. The application of the family study to the prenatal diagnosis is also discussed.     

Pro-453 to Ser mutation in CYP21 is associated with nonclassic steroid 21-hydroxylase deficiency.

Steroid 21-hydroxylase deficiency is the leading cause of impaired cortisol synthesis in congenital adrenal hyperplasia (CAH), with the nonclassic form (NC) comprising approximately 1% of the Caucasian population. The structure of the CYP21 gene was studied in 13 unrelated NC-CAH patients, three affected siblings, and 55 blood donors using polymerase chain reaction. In addition to the Leu-281 and Leu-30 mutations previously associated with NC-CAH, the finding of a Pro-453 to Ser mutation in exon-10 of CYP21 in the NC-CAH patients is reported. Ser-453 was found in 46.2% of unrelated NC-CAH patients, but only 7.7% and 3.6% of salt-wasting CAH patients and blood donors, respectively. In contrast to the Leu-281 and Leu-30 mutations, Ser-453 has not been previously detected in the CYP21 pseudogene (CYP21P) and, therefore, has not likely arisen by gene conversion.     

A 3D model of human P450c21: study of the putative effects of steroid 21-hydroxylase gene mutations

In order to better understand the disease-causing role of missense mutations found in the CYP21 gene from patients affected with congenital adrenal hyperplasia (CAH) due to steroid 21-hydroxylase deficiency, we built two three-dimensional (3D) models of human P450c21 using all known 3D structures of P450s. For each residue affected by a missense mutation, its location in the 3D structure and the putative changes in terms of biochemical properties brought about by the mutation were analyzed. Most of the severe alleles were found to affect residues located in functionally important regions of the molecule such as substrate recognition sites (SRS) or the heme region, whereas moderate mutations were mostly found in less crucial regions of the molecule. Thus, there is a good correlation between the 3D structure study and clinical data and mutagenesis experiments previously reported. In one case, however, the observed clinical severity of the mutation (E380D) did not match its expected severity deduced from the model, pointing to a potential functionally important region of the molecule. Our 3D human models provide a basic model for further studies of mutations responsible for 21-hydroxylase, and for identification of important residues involved in the specific activity of the enzyme.     

Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants

Steroid 21-hydroxylase (cytochrome P450 21A2, CYP21A2) deficiency accounts for ∼95% of individuals with congenital adrenal hyperplasia, a common autosomal recessive metabolic disorder of adrenal steroidogenesis. The effects of amino acid mutations on CYP21A2 activity lead to impairment of the synthesis of cortisol and aldosterone and the excessive production of androgens. In order to understand the structural and molecular basis of this group of diseases, the bovine CYP21A2 crystal structure complexed with the substrate 17-hydroxyprogesterone (17OHP) was determined to 3.0 Å resolution. An intriguing result from this structure is that there are two molecules of 17OHP bound to the enzyme, the distal one being located at the entrance of the substrate access channel and the proximal one bound in the active site. The substrate binding features locate the key substrate recognition residues not only around the heme but also along the substrate access channel. In addition, orientation of the skeleton of the proximal molecule is toward the interior of the enzyme away from the substrate access channel. The 17OHP complex of CYP21A2 provides a good relationship between the crystal structure, clinical data, and genetic mutants documented in the literature, thereby enhancing our understanding of congenital adrenal hyperplasia. In addition, the location of certain CYP21A2 mutations provides general understanding of structure/function relationships in P450s.     

Steroid 21-hydroxylase (P450c21): a new allele and spread of mutations through the pseudogene

Lesions in the gene encoding the adrenal enzyme steroid 21-hydroxylase (P450c21) result in defective adrenal cortisol synthesis, often accompanied by aldosterone deficiency. The symptoms range from severe neonatal disease to inconspicuous symptoms in adulthood depending on the nature of the mutations. The 21-hydroxylase gene is present in close proximity to a highly homologous pseudogene, and both genes show variation in copy number between individuals. For complete DNA sequence characterization, we have applied selective polymerase chain reaction amplification and direct sequencing of all full-length steroid 21-hydroxylase genes present in individuals. Using healthy individuals with only one remaining steroid 21-hydroxylase allele as normal references, a new allele was found in two siblings, in whom clinical and laboratory findings demonstrated moderate enzyme deficiency. Full-length sequencing of this allele displayed an Arg 484 to Pro codon change in exon 10, in the same position as a previously identified GG to C mutation found in a patient with severe 21 -hydroxylase deficiency. Arg 484 is located within a stretch of amino acids that are highly conserved between mammalian 21-hydroxylases. The finding of the presently reported 21-hydroxylase allele indicates that the GG to C mutation from the severely affected patient has arisen by a two-step mechanism, consisting of a G to C transversion accompanied by an adjacent G deletion. When sequencing 26 pseudogenes, both these mutations, which are not present in the pseudogenes hitherto reported, were found at low frequency together with a number of other polymorphisms. Thus, also rare mutations can spread via the pseudogene and can therefore be expected to arise independently in unrelated individuals.     

Molecular biology of rat steroid 11 beta-hydroxylase [P450(11 beta)] and aldosterone synthase [P450(11 beta, aldo)].

The molecular features of rat steroid 11ß-hydroxylase [P450(11ß)] and aldosterone synthase [P450(11ß, aldo)] are discussed. P450(11ß) is biosynthesized as a precursor form composed of 499 amino acids, having a 24-amino acid extension peptide. Two species of P450(11ß, aldo) were identified; a precursor form of P450(11ß, aldo)-1 is 510 amino acids long and has a 34-amino acid extension peptide, while that of P450(11ß, aldo)-2 is 500 amino acids long and has a 24-amino acid extension peptide. The 286th amino acid of P450(11ß, aldo)-1 is Glu, while that of P450(11ß, aldo)-2 is Lys. The cDNA-expression studies showed that P450(11ß, aldo)-1 had the aldosterone producing activity whereas P450(11ß, aldo)-2 had no activity, suggesting that Glu286 of P450(11ß, aldo) plays an important role in the catalysis. The amino acid sequence of a region in P450(11ß) from Leu337 through Pro352 is highly conserved among the steroidogenic P450s. Functional expression studies on the cDNAs for two P450(11ß)s showed that P450(11ß) catalyzes the 11ß-, 18- and 19-hydroxylations of 11-deoxycorticosterone, but not the aldosterone synthesis. P450(11ß, aldo), on the other hand, catalyzes the conversion of 11-deoxycorticosterone to corticosterone, 18-hydroxycorticosterone and aldosterone. The two P450(11ß)s were also shown to catalyze the conversion of 11-deoxycortisol to cortisol, 18-hydroxycortisol and cortisone.     

R339H and P453S: CYP21 mutations associated with nonclassic steroid 21-hydroxylase deficiency that are not apparent gene conversions.

Steroid 21-hydroxylase deficiency is the most common enzymatic defect causing congenital adrenal hyperplasia, an inherited disorder of cortisol biosynthesis. All mutations thus far characterized that cause this disorder appear to result from recombinations between the gene encoding the enzyme, CYP21B (CYP21), and the adjacent pseudogene, CYP21A (CYP21P). These are either deletions caused by unequal crossing-over during meiosis or apparent transfers of deleterious sequences from CYP21A to CYP21B, a phenomenon termed gene conversion. However, a small percentage of alleles do not carry such a mutation. We analyzed DNA from a patient with the mild, nonclassic form of 21-hydroxylase deficiency, who carried one allele that had no gene conversions detectable by hybridization with oligonucleotide probes. Sequence analysis revealed that this allele carried two missense mutations, R339H and P453S, neither of which has been previously observed in CYP21A or CYP21B. Each of these mutations was introduced into CYP21 cDNA which was then expressed in COS1 cells using a vaccinia virus system. Each mutation reduced the ability of the enzyme to 21-hydroxylate 17-hydroxyprogesterone to 50% of normal and the ability to metabolize progesterone to 20% of normal. Thus, each of these mutations represents a potential nonclassic 21-hydroxylase deficiency allele that is not the result of an apparent gene conversion.     

Substitution of Ile-172 to Asn in the steroid 21-hydroxylase B (P450c21B) gene in a Finnish patient with the simple virilizing form of congenital adrenal hyperplasia

Summary The steroid 21-hydroxylase enzyme (P450c21) is a member of the cytochrome P450 gene superfamily and is essential in the synthesis of cortisol and aldosterone. Defects in the P450c21B gene cause congenital adrenal hyperplasia (CAH), a common genetic disorder leading to virilization of newborn females. To avoid the standard cloning of mutant P450c21 genes from genomic libraries, we amplified the full-length genomic P450c21 genes by polymerase chain reaction (PCR). The amplification was followed by cloning and sequencing of a defective P450c21B gene. The strategy described here is generally applicable, thus making a simple characterization of the complete P450c21B gene possible. The method was tested in one patient suffering from the simple virilizing form of CAH. The sequence of three independent clones originating from the defective P450c21B showed that Ile at position 172 in exon 4 was substituted by Asn. The identical mutation also has been found in other patients with CAH.     

Effects of individual mutations in the P-450(C21) pseudogene on the P-450(C21) activity and their distribution in the patient genomes of congenital steroid 21-hydroxylase deficiency

Recent observations have suggested that the pathological mutations in human P-450(C21) deficiency are generated through gene conversion-like events between the functional gene [P-450(21)B] and the pseudogene [P-450(C21)A]. To address this point more extensively, we investigated the effects of the base changes in the A pseudogene on the P-450(21) activity by using the COS cell expression system. In addition to the defective mutations found previously in the pseudogene, four single base changes with amino acid substitutions of Pro(30), Ile(172), Val(282), or Arg(356) were further identified as causing complete [Arg(356)] or partial [Pro(30), Ile(172), and Val(282)] inactivation of P-450(C21). Blot hybridization analysis of patient DNAs using oligonucleotide probes specific for these mutations revealed that the splicing mutation in the 2nd intron was distributed most frequently in both simple-virilizing and salt-wasting forms. The mutation Ile(172) seemed to be frequent in patients with the less severe simple-virilizing form, whereas the mutation Arg(356), together with other most serious mutations reported previously, was preferentially associated with salt-wasting, the most severe form of the disease. In combination with the present results of the effects of various mutations on the P-50(C21) activity, a survey of the distribution of the various mutations in the patient genomes so far reported suggests that the heterogeneous clinical symptoms of this genetic disease are somehow related to the degree of attenuation of the activities of the mutated gene products.     

Organ-specific properties of cytochromes P-450s21 (steroid 21-hydroxylases) of liver and adrenocortical microsomes

Prothrombin, plasminogen, urokinase- and tissue-type plasminogen activators contain homologous structures known as kringles . The kringles correspond to autonomous structural and folding domains which mediate the binding of these multidomain proteins to other proteins. During evolution the different kringles retained the same gross architecture, the kringle -fold, yet diverged to bind different proteins. We show that the amino acid sequences of the type II structures of the gelatin-binding region of fibronectin are homologous with those of the protease- kringles . Prediction of secondary structures revealed a remarkable agreement in the positions of predicted beta-sheets, suggesting that the folding of kringles and type II structures may also be similar. As a corollary of this finding, the disulphide-bridge pattern of type II structures is shown to be homologous to that in kringles . It is noteworthy that protease- kringles and fibronectin type II structures have similar functions inasmuch as they mediate the binding of multidomain proteins to other proteins. It is proposed that the kringles of proteases and type II structures of fibronectin evolved from a common ancestral protein binding module.     

Expression of bovine cytochrome P450c21 and its fused enzymes with yeast NADPH-cytochrome P450 reductase in Saccharomyces cerevisiae

Recombinant plasmids for expression of bovine cytochrome P450c21 (pA gamma 2), both P450c21 and yeast NADPH-cytochrome P450 reductase (pAR gamma 1), P450c21/yeast reductase fused enzymes (pAF gamma R1, pAF gamma R2, and pAF gamma R20), and yeast reductase/P450c21 fused enzymes (pAFR gamma 1 and pAFR gamma 2) were constructed by using expression vector pAAH5. The plasmids were each introduced into the yeast Saccharomyces cerevisiae AH22 cells. The recombinant yeast strains AH22/pA gamma 2 (Y21) and AH22/pAR gamma 1 (Y21R) produced 2-3 X 10(3) molecules of P450c21 per cell. The cultures of both strains converted progesterone and 17 alpha-hydroxyprogesterone into 11-deoxycorticosterone and 11-deoxycortisol, respectively. The 21-hydroxylase activity per cell of the strain Y21R was about three times higher than that of the strain Y21, probably due to overproduction of yeast reductase. The recombinant yeast strains AH22/pAF gamma R1 (Y21RF1), AH22/pAF gamma R2 (Y21RF2), and AH22/pAF gamma R20 (Y21RF20) produced about 1.1-2.0 X 10(4) molecules per cell of the corresponding P450c21/yeast reductase fused enzymes. The specific 21-hydroxylase activity toward 17 alpha-hydroxyprogesterone per cell of the strains Y21RF1, Y21RF2, and Y21RF20 was about 21, 28, and 49 times higher than that of the strain Y21, respectively. Thus, the fused enzymes were superior to P450c21 in the specific activity and in the expression level in the yeast. The Km values for 17 alpha-hydroxyprogesterone of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 0.29, 0.30, 0.67, and 0.65 microM, respectively. The Vmax values of P450c21 in the strains Y21 and Y21R, and of the fused enzymes in the strains Y21RF1 and Y21RF2 were 28, 124, 151, and 222 moles/min.mole P450c21 or fused enzyme, respectively. These results indicated that the fused enzymes showed lower affinity for the substrate, probably due to structural modification and higher reaction rates through efficient intramolecular electron transfer as compared with those of P450c21. While the strain AH22/pAFR gamma 2 (YR21F2) produced about 3 X 10(4) molecules per cell of the reductase/P450c21 fused enzyme, the specific 21-hydroxylase activity of the fused enzyme toward 17 alpha-hydroxyprogesterone was extremely low, suggesting that the structure of the fused enzyme might not be suited for electron transfer in yeast microsomes.     

Expression of human 21-hydroxylase (P450c21) in bacterial and mammalian cells: a system to characterize normal and mutant enzymes

Cytochrome P450c21 (steroid 21-hydroxylase) is a key enzyme in the synthesis of cortisol, whose deficiency is the cause of a common genetic disease, congenital adrenal hyperplasia. We have expressed P450c21 (steroid 21-hydroxylase) in E. coli and mammalian cells. In E. coli, P450c21 cDNA was cloned into a T7 expression vector to produce a large amount of P450c21 fusion protein, which enabled antiserum production. In mammalian cells, a plasmid containing full-length P450c21 cDNA (phc21) was constructed and transfected into COS-1 cells to produce active P450c21, which was detected by immunoblotting and 21-hydroxylase activity assay. This system was used to assay mutations involved in the disease. Ile172 of phc21 corresponding to the site of mutation in some cases of the disease was mutagenized to become Asn, Leu, His, or Gln. Mutant as well as normal P450c21 was produced when their cDNAs were transfected into COS-1 cells. The mutant proteins, however, had greatly reduced 21-hydroxylase activities. Therefore, missense mutation at Ile172 resulted in inactivation of the enzyme, but not in repression of enzyme synthesis. The Leu for Ile substitution at amino acid 172 did not result in partial restoration of enzymatic activity, indicating that hydrophobicity at this residue may not play a role in its function.