ABSTRACT
Deficiency of 21-hydroxylase enzyme (CYP21A2) represents 90% of cases in congenital adrenal hyperplasia (CAH), an autosomal recessive disease caused by defects in cortisol biosynthesis. Computational prediction and functional studies are often the only way to classify variants to understand the links to disease-causing effects. Here we investigated the pathogenicity of uncharacterized variants in the CYP21A2 gene reported in Brazilian and Portuguese populations. Physicochemical alterations, residue conservation, and effect on protein structure were accessed by computational analysis. The enzymatic performance was obtained by functional assay with the wild-type and mutant CYP21A2 proteins expressed in HEK293 cells. Computational analysis showed that p.W202R, p.E352V, and p.R484L have severely impaired the protein structure, while p.P35L, p.L199P, and p.P433L have moderate effects. The p.W202R, p.E352V, p.P433L, and p.R484L variants showed residual 21OH activity consistent with the simple virilizing phenotype. The p.P35L and p.L199P variants showed partial 21OH efficiency associated with the non-classical phenotype. Additionally, p.W202R, p.E352V, and p.R484L also modified the protein expression level. We have determined how the selected CYP21A2 gene mutations affect the 21OH activity through structural and activity alteration contributing to the future diagnosis and management of CYP21A2 deficiency.
Subject(s)
Genetics, Population , Mutation/genetics , Steroid 21-Hydroxylase/genetics , Adolescent , Amino Acid Sequence , Brazil , Child, Preschool , Computer Simulation , Conserved Sequence , Female , Humans , Infant , Kinetics , Male , Models, Molecular , Mutant Proteins/chemistry , Mutant Proteins/genetics , Portugal , Reproducibility of Results , Steroid 21-Hydroxylase/chemistryABSTRACT
We have investigated interactions of galeterone and its pharmacologically active metabolite - 3-keto-Δ4-galeterone (D4G) - with one of the key enzymes of corticosteroid biosynthesis - steroid 21-monooxygenase (CYP21A2). It was shown by absorption spectroscopy that both compounds induce type I spectral changes of CYP21A2. Spectral dissociation constants (KS ) of complexes of CYP21A2 with galeterone or D4G were calculated as 3.1 ± 0.7 µm and 4.6 ± 0.4 µm, respectively. It was predicted by molecular docking that both ligands similarly bind to the active site of CYP21A2. We have revealed using reconstituted monooxygenase system that galeterone is a competitive inhibitor of CYP21A2 with the inhibition constant (Ki ) value of 12 ± 3 µm, while D4G at the concentrations of 10 and 25 µm does not inhibit the enzyme. Summarizing, based on the in vitro analyses we detected inhibition of CYP21A2 by galeterone and lack of the influence of D4G on this enzyme.
Subject(s)
Androstadienes/chemistry , Benzimidazoles/chemistry , Enzyme Inhibitors/chemistry , Steroid 21-Hydroxylase/chemistry , Drug Interactions , Humans , Male , Molecular Docking Simulation , Prostatic Neoplasms/drug therapyABSTRACT
The aim of this project was to investigate the endocrine disrupting effects of three γ-aminobutyric acid type A receptor (GABAAR) agonists, diazepam (DZ), oxazepam (OX) and alprazolam (AL) using the steroidogenic in vitro H295R cell line assay, a recombinant CYP17A1 assay, qPCR analysis and computational modelling. Similar effects for DZ and OX on the steroidogenesis were observed in the H295R experiment at therapeutically relevant concentrations. Progestagens and corticosteroids were increased up to 10 fold and androgens were decreased indicating CYP17A1 lyase inhibition. For DZ the inhibition on both the hydroxylase and lyase was confirmed by the recombinant CYP17A1 assay, whereas OX did not appear to directly affect the recombinant CYP17A1 enzyme. Androgens were decreased when exposing the H295R cells to AL, indicating a CYP17A1 lyase inhibition. However, this was not confirmed by the recombinant CYP17A1 assay but a down-regulation in gene expression was observed for StAR and CYP17A1. The present study showed that the three investigated benzodiazepines (BZDs) are rather potent endocrine disruptors in vitro, exerting endocrine effects close the therapeutic Cmax. Both direct and indirect effects on steroidogenesis were observed, but molecular modelling indicated no direct interactions between the heme group in the steroidogenic CYP enzymes and the unique diazepin structure. In contrast, physicochemical properties such as high log P, structure and molecular weight similar to that of steroids appeared to influence the endocrine disrupting abilities of the investigated pharmaceuticals in vitro. Docking of the three BZDs in CYP17A1 and CYP21A2 confirmed that shape complementarity and hydrophobic effects seem to determine the binding modes.
Subject(s)
Benzodiazepines/chemistry , Endocrine Disruptors/chemistry , Steroid 17-alpha-Hydroxylase/chemistry , Steroid 21-Hydroxylase/chemistry , Steroids/biosynthesis , Adrenal Cortex Hormones/chemistry , Adrenal Cortex Hormones/pharmacology , Adrenal Glands/drug effects , Alprazolam/chemistry , Alprazolam/pharmacology , Androgens/genetics , Benzodiazepines/pharmacology , Diazepam/chemistry , Diazepam/pharmacology , Endocrine Disruptors/pharmacology , Humans , Molecular Docking Simulation , Oxazepam/chemistry , Oxazepam/pharmacology , Receptors, Androgen/chemistry , Receptors, Androgen/genetics , Receptors, GABA-A/chemistry , Receptors, GABA-A/genetics , Steroid 17-alpha-Hydroxylase/antagonists & inhibitors , Steroid 17-alpha-Hydroxylase/genetics , Steroid 21-Hydroxylase/antagonists & inhibitors , Steroid 21-Hydroxylase/genetics , Steroids/chemistryABSTRACT
Classical congenital adrenal hyperplasia (CAH) caused by pathogenic variants in the steroid 21-hydroxylase gene (CYP21A2) is a severe life-threatening condition. We present a detailed investigation of the molecular and functional characteristics of a novel pathogenic variant in this gene. The patient, 46 XX newborn, was diagnosed with classical salt wasting CAH in the neonatal period after initially presenting with ambiguous genitalia. Multiplex ligation-dependent probe analysis demonstrated a full deletion of the paternal CYP21A2 gene, and Sanger sequencing revealed a novel de novo CYP21A2 variant c.694-696del (E232del) in the other allele. This variant resulted in the deletion of a non-conserved single amino acid, and its functional relevance was initially undetermined. We used both in silico and in vitro methods to determine the mechanistic significance of this mutation. Computational analysis relied on the solved structure of the protein (Protein-data-bank ID 4Y8W), structure prediction of the mutated protein, evolutionary analysis, and manual inspection. We predicted impaired stability and functionality of the protein due to a rotatory disposition of amino acids in positions downstream of the deletion. In vitro biochemical evaluation of enzymatic activity supported these predictions, demonstrating reduced protein levels to 22% compared to the wild-type form and decreased hydroxylase activity to 1-4%. This case demonstrates the potential of combining in-silico analysis based on evolutionary information and structure prediction with biochemical studies. This approach can be used to investigate other genetic variants to understand their potential effects.
Subject(s)
Computer Simulation , Mutation/genetics , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/genetics , Child, Preschool , Evolution, Molecular , Female , Humans , Infant , Infant, NewbornABSTRACT
Congenital adrenal hyperplasia (CAH) is one of the most frequent inborn errors of metabolism, inherited as an autosomal recessive trait. Above 95% of CAH cases are caused by mutations in cytochrome P450 21A2 (CYP21A2). It is a pity that how these mutations affect the structural characteristics and substrate binding of CYP21A2 is still unclear. To this end, molecular dynamics (MD) simulations and binding free energy calculations are performed to investigate the effects of single point mutations (L108R, G292C, G292S, G293D, and T296N) in CYP21A2. The results indicate that mutations could cause the local conformational changes of CYP21A2, affecting the substrate binding by changing the interaction between the protein and heme, changing the charge environment of residues, or introducing steric hindrance. In addition, our work gives a wonderful explanation of the phenomenon that though the substrate binding ability increases, the reaction activity decreases in T296N. The present study provides detailed atomistic insights into the structure-function relationships of CYP21A2, which could contribute to further understanding about 21-hydroxylase deficiency and also provide a theoretical basis for CAH prediction and treatment.
Subject(s)
Mutation , Steroid 21-Hydroxylase , Adrenal Hyperplasia, Congenital/enzymology , Adrenal Hyperplasia, Congenital/genetics , Adrenal Hyperplasia, Congenital/physiopathology , Heme/metabolism , Humans , Molecular Dynamics Simulation , Protein Binding/genetics , Protein Structure, Tertiary/genetics , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/genetics , Steroid 21-Hydroxylase/metabolismABSTRACT
Synthetic glucocorticoids such as methylprednisolone are compounds of fundamental interest to the pharmaceutical industry as their modifications within the sterane scaffold lead to higher inflammatory potency and reduced side effects compared with their parent compound cortisol. In methylprednisolone production, the complex chemical hydroxylation of its precursor medrane in position C21 exhibits poor stereo- and regioselectivity making the process unprofitable and unsustainable. By contrast, the use of a recombinant E. coli system has recently shown high suitability and efficiency. In this study, we aim to overcome limitations in this biotechnological medrane conversion yielding the essential methylprednisolone-precursor premedrol by optimizing the CYP21A2-based whole-cell system on a laboratory scale. We successfully improved the whole-cell process in terms of premedrol production by (a) improving the electron supply to CYP21A2; here we use the N-terminally truncated version of the bovine NADPH-dependent cytochrome P450 reductase (bCPR-27 ) and coexpression of microsomal cytochrome b5 ; (b) enhancing substrate access to the heme by modification of the CYP21A2 substrate access channel; and (c) circumventing substrate inhibition which is presumed to be the main limiting factor of the presented system by developing an improved fed-batch protocol. By overcoming the presented limitations in whole-cell biotransformation, we were able to achieve a more than 100% improvement over the next best system under equal conditions resulting in 691 mg·L-1 ·d-1 premedrol.
Subject(s)
Escherichia coli/genetics , Metabolic Engineering/methods , Methylprednisolone , Recombinant Proteins/metabolism , Steroid 21-Hydroxylase/metabolism , Animals , Biotransformation , Cattle , Escherichia coli/metabolism , Hydroxylation , Methylprednisolone/analogs & derivatives , Methylprednisolone/analysis , Methylprednisolone/chemistry , Methylprednisolone/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/geneticsABSTRACT
OBJECTIVE: Congenital adrenal hyperplasia (CAH) is an inborn error of metabolism and a common disorder of sex development where >90% of all cases are due to 21-hydroxylase deficiency. Novel and rare pathogenic variants account for 5% of all clinical cases. Here, we sought to investigate the functional and structural effects of four novel (p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro) and three combinations of CYP21A2 variants (i.e. one allele containing two variants p.[Ile172Asn;Val358Ile], p.[Val281Leu;Arg369Gln], or p.[Asp377Tyr;Leu461Pro]) identified in patients with CAH. METHODS: All variants were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells and enzyme activities directed toward the two natural substrates (17-hydroxyprogesterone and progesterone) were determined. In parallel, in silico prediction of the pathogenicity of the variants based on the human CYP21 X-ray structure was performed. RESULTS: The novel variants, p.Val358Ile, p.Arg369Gln, p.Asp377Tyr, and p.Leu461Pro exhibited residual enzymatic activities within the range of non-classic (NC) CAH variants (40-82%). An additive effect on the reduction of enzymatic activity (1-17%) was observed when two variants were expressed together, as identified in several patients, resulting in either NC or more severe phenotypes. In silico predictions were in line with the in vitro data except for p.Leu461Pro. CONCLUSIONS: Altogether, the combination of clinical data, in silico prediction, and data from in vitro studies are important for establishing a correct genotype and phenotype correlation in patients with CAH.
Subject(s)
Adrenal Hyperplasia, Congenital , Alleles , Models, Molecular , Mutation, Missense , Steroid 21-Hydroxylase , Adolescent , Adrenal Hyperplasia, Congenital/enzymology , Adrenal Hyperplasia, Congenital/genetics , Adult , Amino Acid Substitution , Animals , COS Cells , Child , Child, Preschool , Chlorocebus aethiops , Female , Humans , Infant , Male , Protein Domains , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/geneticsABSTRACT
Human cytochrome P450 enzymes are membrane-bound heme-containing monooxygenases. As is the case for many heme-containing enzymes, substitution of the metal in the center of the heme can be useful for mechanistic and structural studies of P450 enzymes. For many heme proteins, the iron protoporphyrin prosthetic group can be extracted and replaced with protoporphyrin containing another metal, but human membrane P450 enzymes are not stable enough for this approach. The method reported herein was developed to endogenously produce human membrane P450 proteins with a nonnative metal in the heme. This approach involved coexpression of the P450 of interest, a heme uptake system, and a chaperone in Escherichia coli growing in iron-depleted minimal medium supplemented with the desired trans-metallated protoporphyrin. Using the steroidogenic P450 enzymes CYP17A1 and CYP21A2 and the drug-metabolizing CYP3A4, we demonstrate that this approach can be used with several human P450 enzymes and several different metals, resulting in fully folded proteins appropriate for mechanistic, functional, and structural studies including solution NMR.
Subject(s)
Cytochrome P-450 CYP3A/metabolism , Metalloporphyrins/metabolism , Metals/metabolism , Protoporphyrins/metabolism , Steroid 17-alpha-Hydroxylase/metabolism , Steroid 21-Hydroxylase/metabolism , Cytochrome P-450 CYP3A/chemistry , Humans , Metalloporphyrins/chemistry , Protein Folding , Protoporphyrins/chemistry , Steroid 17-alpha-Hydroxylase/chemistry , Steroid 21-Hydroxylase/chemistryABSTRACT
Inhibition of androgen biosynthesis is clinically effective for treating androgen-responsive prostate cancer. Abiraterone is a clinical first-in-class inhibitor of cytochrome P450 17A1 (CYP17A1) required for androgen biosynthesis. However, abiraterone also causes hypertension, hypokalemia, and edema, likely due in part to off-target inhibition of another steroidogenic cytochrome P450, CYP21A2. Abiraterone analogs were designed based on structural evidence that B-ring substituents may favorably interact with polar residues in binding CYP17A1 and sterically clash with residues in the CYP21A2 active site. The best analogs increased selectivity of CYP17A1 inhibition up to 84-fold compared with 6.6-fold for abiraterone. Cocrystallization with CYP17A1 validated the intended new contacts with CYP17A1 active site residues. Docking these analogs into CYP21A2 identified steric clashes that likely underlie decreased binding and CYP21A2 inhibition. Overall, these analogs may offer a clinical advantage in the form of reduced side effects.
Subject(s)
Androstenes/chemistry , Androstenes/pharmacology , Cytochrome P-450 Enzyme Inhibitors/chemistry , Cytochrome P-450 Enzyme Inhibitors/pharmacology , Drug Design , Steroid 17-alpha-Hydroxylase/antagonists & inhibitors , Steroid 21-Hydroxylase/antagonists & inhibitors , Androstenes/metabolism , Catalytic Domain , Cytochrome P-450 Enzyme Inhibitors/metabolism , Humans , Molecular Docking Simulation , Steroid 17-alpha-Hydroxylase/chemistry , Steroid 17-alpha-Hydroxylase/metabolism , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/metabolismABSTRACT
Steroid 21-Hydroxylase deficiency is an inherited autosomal recessive metabolic disorder of the adrenal steroidogenesis caused due to mutations in the CYP21A2 gene in 95% of CAH cases. Notably, the de novo mutations arise at the rate of 3-5%, therefore the functional characterization is of utmost importance for categorization of the novel mutations. Herein, we have functionally characterized the CYP21A2 missense mutations viz., p. F306V and p. H365N. Notably, both the mutations were harbored by the patients exhibiting the non classical phenotype. We followed the approach of in vitro characterization of the mutant proteins expressed in COS7 cells. Of note, all the mutant constructs exhibited reduced residual enzyme activity fraternized with altered kinetic constants accompanied by higher requirement for the activation energy. Further, there was reduced protein expression in the mutant constructs to that of the wild-type. Molecular modeling suggested alteration in the structure-function relationship of the protein due to mutations. The evidence suggested by the in vitro and the in silico characterization of mutations directed us to conclude that both, p. F306V and p. H365N should be considered as non classical CAH causing mutations. Conceivably, the knowledge about the functional consequences of the mutations is the basis for improved genetic counseling with respect to prognosis and therapeutic implications.
Subject(s)
Adrenal Hyperplasia, Congenital/genetics , Models, Molecular , Mutant Proteins/genetics , Steroid 21-Hydroxylase/genetics , Adolescent , Adrenal Hyperplasia, Congenital/pathology , Adult , Animals , COS Cells , Child , Chlorocebus aethiops , Female , Gene Expression Regulation , Genotype , Humans , Male , Mutant Proteins/chemistry , Mutation, Missense , Phenotype , Steroid 21-Hydroxylase/chemistry , Young AdultABSTRACT
Abiraterone is an inhibitor of CYP17A1 which is used for the treatment of castration resistant prostate cancer. Abiraterone is known to inhibit several drug metabolizing cytochrome P450 enzymes including CYP1A2, CYP2D6, CYP2C8, CYP2C9, CYP2C19, CYP3A4 and CYP3A5, but its effects on steroid metabolizing P450 enzymes are not clear. In preliminary results, we had observed inhibition of CYP21A2 by 1µM abiraterone. Here we are reporting the effect of abiraterone on activities of CYP21A2 in human adrenal cells as well as with purified recombinant CYP21A2. Cells were treated with varying concentrations of abiraterone for 24h and CYP21A2 activity was measured using [3H] 17-hydroxyprogesterone as substrate. Whole steroid profile changes were determined by gas chromatography-mass spectrometry. Binding of abiraterone to purified CYP21A2 protein was measured spectroscopically. Computational docking was used to study the binding and interaction of abiraterone with CYP21A2. Abiraterone caused significant reduction in CYP21A2 activity in assays with cells and an inhibition of CYP21A2 activity was also observed in experiments using recombinant purified proteins. Abiraterone binds to CYP21A2 with an estimated Kd of 6.3µM. These inhibitory effects of abiraterone are at clinically used concentrations. A loss of CYP21A2 activity in combination with reduction of CYP17A1 activities by abiraterone could result in lower cortisol levels and may require monitoring for any potential adverse effects.
Subject(s)
Androstenes/pharmacology , Antineoplastic Agents/pharmacology , Steroid 21-Hydroxylase/antagonists & inhibitors , Cell Line, Tumor , Escherichia coli/genetics , Humans , Male , Molecular Docking Simulation , Prostatic Neoplasms , Steroid 17-alpha-Hydroxylase/antagonists & inhibitors , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/genetics , Steroid 21-Hydroxylase/metabolism , Steroids/metabolismABSTRACT
Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17α-hydroxyprogesterone (17α-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure of WT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17α-OH-progesterone). We found that the 17α-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either salt-wasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37-13,000-fold higher than for WT P450 21A2. Cytochrome b5, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (≥ 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3-progesterone, indicating that C-H bond breaking is a rate-limiting step over a 104-fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insight into the effects of disease-causing mutations on this important enzyme.
Subject(s)
Adrenal Hyperplasia, Congenital/enzymology , Mutation , Steroid 21-Hydroxylase/chemistry , Adrenal Hyperplasia, Congenital/genetics , Circular Dichroism , Cytochromes b5/chemistry , Cytochromes b5/genetics , Cytochromes b5/metabolism , Deuterium Exchange Measurement , Enzyme Stability , Hot Temperature , Humans , Protein Domains , Spectrophotometry, Ultraviolet , Steroid 21-Hydroxylase/genetics , Steroid 21-Hydroxylase/metabolismABSTRACT
Congenital adrenal hyperplasia due to 21-hydroxylase deficiency accounts for 90-95% of CAH cases. In this work we performed an extensive survey of mutations and SNPs modifying the coding sequence of the CYP21A2 gene. Using bioinformatic tools and two plausible CYP21A2 structures as templates, we initially classified all known mutants (n = 343) according to their putative functional impacts, which were either reported in the literature or inferred from structural models. We then performed a detailed analysis on the subset of mutations believed to exclusively impact protein stability. For those mutants, the predicted stability was calculated and correlated with the variant's expected activity. A high concordance was obtained when comparing our predictions with available in vitro residual activities and/or the patient's phenotype. The predicted stability and derived activity of all reported mutations and SNPs lacking functional assays (n = 108) were assessed. As expected, most of the SNPs (52/76) showed no biological implications. Moreover, this approach was applied to evaluate the putative synergy that could emerge when two mutations occurred in cis. In addition, we propose a putative pathogenic effect of five novel mutations, p.L107Q, p.L122R, p.R132H, p.P335L and p.H466fs, found in 21-hydroxylase deficient patients of our cohort.
Subject(s)
Adrenal Hyperplasia, Congenital/genetics , Genetic Variation , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/genetics , Computer Simulation , Humans , Models, Molecular , Mutation , Polymorphism, Single Nucleotide , Protein Conformation , Protein Stability , Steroid 21-Hydroxylase/metabolism , Structure-Activity RelationshipABSTRACT
Congenital adrenal hyperplasia (CAH) owing to 21-hydroxylase deficiency is caused by the autosomal recessive inheritance of mutations in the gene CYP21A2. CYP21A2 mutations lead to variable impairment of the 21-hydroxylase enzyme, which, in turn, is associated with three clinical phenotypes, namely, salt wasting, simple virilizing, and nonclassical CAH. However, it is known that a given mutation can associate with different clinical phenotypes, resulting in a high rate of genotype-phenotype nonconcordance. We aimed to study the genotype-phenotype nonconcordance in a family with three siblings affected with nonclassical CAH. All had hormonal evidence of nonclassical CAH, but this phenotype could not be explained by the genotype obtained from commercial CYP21A2 genetic testing, which revealed heterozygosity for the maternal 30 kb deletion mutation. We performed Sanger sequencing of the entire CYP21A2 gene in this family to search for a rare mutation that was not covered by commercial testing and found in the three siblings a second, rare c.1097G>A (p.R366H) mutation in exon 8. Computational modeling confirmed that this was a mild mutation consistent with nonclassical CAH. We recommend that sequencing of entire genes for rare mutations should be carried out when genotype-phenotype nonconcordance is observed in patients with autosomal recessive monogenic disorders, including CAH.
Subject(s)
Adrenal Hyperplasia, Congenital/genetics , Exons , Gene Deletion , Heterozygote , Models, Molecular , Point Mutation , Steroid 21-Hydroxylase/genetics , Adrenal Glands/drug effects , Adrenal Glands/metabolism , Adrenal Glands/physiopathology , Adrenal Hyperplasia, Congenital/drug therapy , Adrenal Hyperplasia, Congenital/metabolism , Adrenal Hyperplasia, Congenital/physiopathology , Amino Acid Substitution , Aromatase Inhibitors/therapeutic use , Child , Computational Biology , DNA Mutational Analysis , Drug Therapy, Combination , Expert Systems , Glucocorticoids/therapeutic use , Humans , Male , Pedigree , Protein Conformation , Siblings , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/metabolism , Treatment OutcomeABSTRACT
Cytochrome P450 21A2 is a key player in steroid 21-hydroxylation and converts progesterone to 11-deoxycorticosterone and 17α-hydroxy progesterone to 11-deoxycortisol. More than 100 mutations in P450 21A2 have been established in patients thus far; these account for the vast majority of occurrences of congenital adrenal hyperplasia (CAH), which is among the most common heritable metabolic diseases in humans. CAH phenotypes range from the most severe, salt-wasting (SW), to the simple virilizing (SV), and nonclassical (NC) CAH forms. We recently determined the crystal structure of human P450 21A2 in complex with progesterone. To gain more insight into the structural and stability changes underlying the phenotypes of individual mutations, we analyzed 24 SW, SV, and NC mutants in the context of the crystal structure of the human enzyme. Our analysis reveals clear differences in the localization of SW, SV, and NC mutations, with many of the first type mapping to the active site and near the heme and/or substrate and mostly resulting in complete loss of enzyme activity. Conversely, NC mutations are often found near the periphery and close to the surface of the protein, and mutant enzymes retain partial activity. The main conclusion from the mutation-structure-activity study is that the severity of the CAH clinical manifestations can be directly correlated with the degree of mutation-induced damage in terms of protein fold stability and active site changes in the structural model. Thus, the NC phenotype is typically associated with mutations that have a compensatory effect, ie, H-bonding replacing hydrophobic interactions and vice versa.
Subject(s)
Adrenal Hyperplasia, Congenital/genetics , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/genetics , Crystallography, X-Ray , Humans , Mutation/genetics , Progesterone/metabolism , Protein Folding , Steroid 21-Hydroxylase/metabolism , Structure-Activity RelationshipABSTRACT
Cytochrome P450 (P450) 21A2 is the major steroid 21-hydroxylase, and deficiency of this enzyme is involved in â¼95% of cases of human congenital adrenal hyperplasia, a disorder of adrenal steroidogenesis. A structure of the bovine enzyme that we published previously (Zhao, B., Lei, L., Kagawa, N., Sundaramoorthy, M., Banerjee, S., Nagy, L. D., Guengerich, F. P., and Waterman, M. R. (2012) Three-dimensional structure of steroid 21-hydroxylase (cytochrome P450 21A2) with two substrates reveals locations of disease-associated variants. J. Biol. Chem. 287, 10613-10622), containing two molecules of the substrate 17α-hydroxyprogesterone, has been used as a template for understanding genetic deficiencies. We have now obtained a crystal structure of human P450 21A2 in complex with progesterone, a substrate in adrenal 21-hydroxylation. Substrate binding and release were fast for human P450 21A2 with both substrates, and pre-steady-state kinetics showed a partial burst but only with progesterone as substrate and not 17α-hydroxyprogesterone. High intermolecular non-competitive kinetic deuterium isotope effects on both kcat and kcat/Km, from 5 to 11, were observed with both substrates, indicative of rate-limiting C-H bond cleavage and suggesting that the juxtaposition of the C21 carbon in the active site is critical for efficient oxidation. The estimated rate of binding of the substrate progesterone (kon 2.4 × 10(7) M(-1) s(-1)) is only â¼2-fold greater than the catalytic efficiency (kcat/Km = 1.3 × 10(7) M(-1) s(-1)) with this substrate, suggesting that the rate of substrate binding may also be partially rate-limiting. The structure of the human P450 21A2-substrate complex provides direct insight into mechanistic effects of genetic variants.
Subject(s)
Progesterone/chemistry , Progesterone/metabolism , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/metabolism , Amino Acid Sequence , Animals , Binding Sites , Catalysis , Catalytic Domain , Cattle , Crystallography, X-Ray , Deuterium/chemistry , Humans , Hydroxylation , Kinetics , Molecular Sequence Data , Oxidation-Reduction , Protein Binding , Protein Conformation , Sequence Homology, Amino Acid , Substrate SpecificityABSTRACT
Congenital adrenal hyperplasia (CAH) due to 21-hydroxylase deficiency is caused by mutations in the CYP21A2 gene. The residual enzyme activity is strongly associated with the phenotype. We describe a rare case of CAH with a rare CYP21A2 mutation. The patient was a one-year-old Japanese boy. At 16 days old, he was referred to our hospital because of elevated serum 17-OH-progesterone (17-OHP) levels in neonatal screening. The compound heterozygous mutations (IVS2-13 A/C>G, and p.E431K) in CYP21A2 were identified at 2 months old, and we diagnosed non-classical CAH, since he did not have significant physical signs (pigmentation and salt-wasting). However, his body weight decreased, and his serum 17-OHP level (99.5 ng/mL) was elevated at 3 months old. Steroid replacement therapy was started at 3 months old. Our patient's clinical course resembled simple virilizing (SV) CAH, but classification was difficult because the patient showed increased renin activity indicating an aldosterone deficiency, and late onset of symptoms. While the IVS 2-13 A/C>G mutation is common in the classical form of CAH, p.E431K is a rare point mutation. Functional analysis revealed that the residual enzyme activity of p.E431L was 5.08±2.55% for 17-OHP and 4.12±2.37% for progesterone, which is consistent with SV CAH. p.E431 is localized in the L-helix near the heme-binding site. The mutation might interfere with heme binding, leading to deactivation of CYP21A2. This report showed that CYP21A2 p.E431 has an important effect on enzyme activity.
Subject(s)
Adrenal Hyperplasia, Congenital/genetics , Heterozygote , Point Mutation , Steroid 21-Hydroxylase/genetics , 17-alpha-Hydroxyprogesterone/blood , 17-alpha-Hydroxyprogesterone/metabolism , Adrenal Hyperplasia, Congenital/blood , Adrenal Hyperplasia, Congenital/drug therapy , Adrenal Hyperplasia, Congenital/enzymology , Amino Acid Substitution , Anti-Inflammatory Agents/therapeutic use , Binding, Competitive , Family Health , Fludrocortisone/therapeutic use , Heme/metabolism , Humans , Hydrocortisone/therapeutic use , Infant , Male , Parents , Progesterone/metabolism , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/metabolism , Substrate Specificity , Treatment Outcome , Up-Regulation/drug effectsABSTRACT
Some cytochrome P450 enzymes epoxidize unsaturated substrates, but this activity has not been described for the steroid hydroxylases. Physiologic steroid substrates, however, lack carbon-carbon double bonds in the parts of the pregnane molecules where steroidogenic hydroxylations occur. Limited data on the reactivity of steroidogenic P450s toward olefinic substrates exist, and the study of occult activities toward alternative substrates is a fundamental aspect of the growing field of combinatorial biosynthesis. We reasoned that human P450c17 (steroid 17-hydroxylase/17,20-lyase, CYP17A1), which 17- and 16α-hydroxylates progesterone, might catalyze the formation of the 16α,17-epoxide from 16,17-dehydroprogesterone (pregna-4,16-diene-3,20-dione). CYP17A1 catalyzed the novel 16α,17-epoxidation and the ordinarily minor 21-hydroxylation of 16,17-dehydroprogesterone in a 1:1 ratio. CYP17A1 mutation A105L, which has reduced progesterone 16α-hydroxylase activity, gave a 1:5 ratio of epoxide:21-hydroxylated products. In contrast, human P450c21 (steroid 21-hydroxylase, CYP21A2) converted 16,17-dehydroprogesterone to the 21-hydroxylated product and only a trace of epoxide. CYP21A2 mutation V359A, which has significant 16α-hydroxylase activity, likewise afforded the 21-hydroxylated product and slightly more epoxide. CYP17A1 wild-type and mutation A105L do not 21- or 16α-hydroxylate pregnenolone, but the enzymes 21-hydroxylated and 16α,17-epoxidized 16,17-dehydropregnenolone (pregna-5,16-diene-3ß-ol-20-one) in 4:1 or 12:1 ratios, respectively. Catalase and superoxide dismutase did not prevent epoxide formation. The progesterone epoxide was not a time-dependent, irreversible CYP17A1 inhibitor. Our substrate modification studies have revealed occult epoxidase and 21-hydroxylase activities of CYP17A1, and the fraction of epoxide formed correlated with the 16α-hydroxylase activity of the enzymes.
Subject(s)
Steroid 17-alpha-Hydroxylase/chemistry , Steroid 17-alpha-Hydroxylase/metabolism , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/metabolism , Amino Acid Substitution , Epoxy Compounds/chemistry , Epoxy Compounds/metabolism , Humans , Hydroxylation , Kinetics , Magnetic Resonance Spectroscopy , Molecular Structure , Mutagenesis, Site-Directed , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Steroid 17-alpha-Hydroxylase/genetics , Steroid 21-Hydroxylase/genetics , Steroids/chemistry , Steroids/metabolism , Substrate SpecificityABSTRACT
Congenital adrenal hyperplasia (CAH) is an autosomal recessive disorder mainly caused by defects in the 21-hydroxylase gene (CYP21A2), coding for the enzyme 21-hydroxylase (21-OH). About 95% of the mutations arise from gene conversion between CYP21A2 and the inactive pseudogene CYP21A1P: only 5% are novel CYP21A2 mutations, in which functional analysis of mutant enzymes has been helpful to correlate genotype-phenotype. In the present study, we describe 3 novel point mutations (p.L122P, p.Q481X, and p.E161X) in 3 Italian patients with CAH: the fourth mutation (p.M150R) was found in the carrier state. Molecular modeling suggests a major impact on 21-hydroxylase activity, and functional analysis after expression in COS-7 cells confirms reduced enzymatic activity of the mutant enzymes. Only the p.M150R mutation affected the activity to a minor extent, associated with NC CAH. CYP21A2 genotyping and functional characterization of each disease-causing mutation has relevance both for treatment and genetic counseling to the patients.
Subject(s)
Adrenal Hyperplasia, Congenital/enzymology , Adrenal Hyperplasia, Congenital/genetics , Mutation/genetics , Steroid 21-Hydroxylase/chemistry , Steroid 21-Hydroxylase/genetics , Amino Acid Sequence , Animals , Blotting, Western , COS Cells , Child , Chlorocebus aethiops , Female , Genotype , Humans , Infant, Newborn , Italy , Male , Molecular Sequence Data , Mutant Proteins/metabolism , Phenotype , Protein Structure, Secondary , Sequence AlignmentABSTRACT
CYP17 (steroid 17α-hydroxylase/17,20-lyase) is a key enzyme in steroid hormone biosynthesis. It catalyzes two independent reactions at the same active center and has a unique ability to differentiate Δ(4)-steroids and Δ(5)-steroids in the 17,20-lyase reaction. The present work presents a complex experimental analysis of the role of CYP17 in the metabolism of 7-dehydrosteroids. The data indicate the existence of a possible alternative pathway of steroid hormone biosynthesis using 7-dehydrosteroids. The major reaction products of CYP17 catalyzed hydroxylation of 7-dehydropregnenolone have been identified. Catalytic activity of CYP17 from different species with 7-dehydropregnenolone has been estimated. It is shown that CYP21 cannot use Δ(5)-Δ(7) steroids as a substrate.