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Journal of Clinical Endocrinology & Metabolism , doi:10.1210/jc.2005-0089
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The Journal of Clinical Endocrinology & Metabolism Vol. 90, No. 6 3724-3730
Copyright © 2005 by The Endocrine Society

Congenital Adrenal Hyperplasia Due to 11-Hydroxylase Deficiency: Functional Characterization of Two Novel Point Mutations and a Three-Base Pair Deletion in the CYP11B1 Gene

Nils Krone, Felix G. Riepe, Dorothea Götze, Eckhard Korsch, Manfred Rister, Jens Commentz, Carl-Joachim Partsch, Joachim Grötzinger, Michael Peter and Wolfgang G. Sippell

Division of Pediatric Endocrinology (N.K., F.G.R., D.G., C.-J.P., M.P., W.G.S.), Department of Pediatrics, Christian-Albrechts-Universität zu Kiel, Universitätsklinikum Schleswig-Holstein (Campus Kiel), D-24105 Kiel, Germany; Kinderkrankenhaus der Stadt Köln (E.K.), D-50735 Köln, Germany; Klinik für Kinder-und Jugendmedizin (M.R.), Klinikum Kemperhof, D-56073 Koblenz, Germany; Medical Practice for Pediatric Endocrinology (J.C.), D-22767 Hamburg, Germany; and Biochemisches Institut (J.G.), Christian-Albrechts-Universität zu Kiel, D-24098 Kiel, Germany

Address all correspondence and requests for reprints to: Wolfgang G. Sippell, M.D., Professor of Pediatrics, Division of Pediatric Endocrinology, Department of Pediatrics, Christian-Albrechts-Universität zu Kiel, Universitätskinderklinik, Schwanenweg 20, D-24105 Kiel, Germany. E-mail: sippell{at}pediatrics.uni-kiel.de.


   Abstract
Top
 Abstract
Introduction
 Patients and Methods
 Results
 Discussion
 References
 
Congenital adrenal hyperplasia is a group of autosomal recessive disorders second most often caused by deficiency of steroid 11-hydroxylase (CYP11B1) due to mutations in the CYP11B1 gene. We studied the functional and structural consequences of two novel missense mutations (W116C, L299P) and an in-frame 3-bp deletion ({Delta}F438) in the CYP11B gene, detected in three unrelated families. All patients are suffering from classical CYP11B1 deficiency. In vitro expression studies in COS-7 cells revealed a decreased CYP11B1 activity in the W116C mutant to 2.9 ± 0.9% (SD) for the conversion of 11-deoxycortisol to cortisol. The L299P mutant reduced the enzymatic activity to 1.2 ± 0.9%, whereas the {Delta}F438 mutation resulted in no measurable residual CYP11B1 activity. Introduction of these mutations in a three-dimensional model structure of the CYP11B1 protein provides a possible explanation for the in vitro measured effects. We hypothesize that the W116C mutation influences the conformational change of the 11-hydroxylase protein necessary for substrate access and product release. The L299P mutation causes a change in the position of the I helix relative to the heme group, whereas the {Delta}F438 mutation results in a steric disarrangement of the heme group relative to the enzyme. Studying the enzyme function in vitro helps to understand the phenotypical expression and disease severity of 11-hydroxylase deficiency, which is the basis for accurate genetic counseling, prenatal diagnosis, and treatment. Moreover, the combination of in vitro enzyme function and molecular modeling provides new insights in cytochrome P450 structural-functional relationships.


   Introduction
Top
 Abstract
 Introduction
Patients and Methods
 Results
 Discussion
 References
 
CONGENITAL ADRENAL hyperplasia (CAH) ranks among the most frequent inborn errors of metabolism following an autosomal recessive trait. It is caused by the loss or severe decrease in activity in one of the five steroidogenic enzymes involved in cortisol biosynthesis. Approximately 90–95% of all cases are due to steroid 21-hydroxylase deficiency (21OHD) (1, 2, 3), and about 5–8% are caused by 11-hydroxylase deficiency (11OHD) (4) and thus occur in approximately 1 to 200,000 live births (2, 5, 6). A high incidence has been reported in Israel among Jewish immigrants from Morocco, a relatively inbred population, with a disease frequency of about 1 in 5000 to 1 in 7000 live births (7).

The deficiency of the 11-hydroxylase (CYP11B1) results in decreased cortisol secretion, elevated plasma levels of ACTH, and accumulation of steroid precursors. These are shunted into the androgen synthesis pathway, leading to hyperandrogenism. Accumulation of 11-deoxycorticosterone and its metabolites causes hypertension in about two thirds of patients. Phenotypical expression of classic 11OHD leads to virilization of external genitalia in newborn females and precocious pseudopuberty combined with rapid somatic growth and bone age acceleration, due to reactive androgen overproduction in both sexes. Female patients with nonclassic CYP11B1 deficiency are born with normal genitalia and present with signs of androgen excess as children. Women may present with hirsutism and oligomenorrhea in adulthood. However, only a minor percentage of women with hirsutism and oligomenorrhea suffer from nonclassic 11OHD (8, 9).

The 11-hydroxylase gene (CYP11B1) is located on chromosome 8q22, approximately 40 kb from the highly homologous CYP11B2 gene encoding for the aldosterone synthase (10). CYP11B1-inactivating mutations have been shown to be distributed over the entire coding region, whereas a cluster is reported in exons 2, 6, 7, and 8 (11, 12). Mutations causing classic (11, 12, 13, 14, 15, 16, 17, 18, 19, 20) and nonclassic (9) CYP11B1 deficiency have been identified.

In this study, we performed the molecular genetic analysis of the CYP11B1 gene in three unrelated families with patients suffering from classic 11OHD. Sequence analysis led to the detection of three novel mutations. The functional analysis of the two missense and one in-frame 3-bp deletion revealed a severe or complete loss of 11-hydroxylase activity of the mutant CYP11B1 proteins proving the pathogenicity of the detected mutations. The in vitro measured alterations of the 11-hydroxylase mutants’ enzymatic activity could be rationalized with the help of a three-dimensional molecular model indicating putative structural change in the protein structure and providing further insights into the function of cytochrome P450 enzymes.


   Patients and Methods
Top
 Abstract
 Introduction
 Patients and Methods
Results
 Discussion
 References
 
Patients, clinical presentation, and hormonal analyses

Case A.T. The female patient (46, XX) was born at term to healthy parents of Turkish origin, who are first-degree cousins. She showed ambiguous external genitalia (Prader genital stage III) and had conspicuously elevated 17-hydroxyprogesterone levels (50 nmol/liter; normal < 20 nmol/liter) in the CAH newborn screening. She was referred at the age of 14 yr to a pediatric endocrinologist (E.K.) for further diagnostics and treatment. There was no clinical evidence for hypertension. Ultrasound and x-ray examinations showed Mullerian structures and normal adrenals. Further diagnostics revealed a situs inversus totalis including dextrocardia and an interrupted inferior vena cava with vena azygos continuation. Multisteroid analysis (21) after a short ACTH test revealed the typical pattern for CAH due to 11OHD (Table 1Go). Plasma testosterone 1.06 nmol/liter (normal range 0.15–0.48 nmol/liter) and androstenedione 13.68 nmol/liter (normal range 0.49–4.92 nmol/liter) were also highly elevated.


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  		TABLE 1. Results of multisteroid analysis at diagnosis/before treatment

 
Case M.M. The male patient originates from Iraq and was referred under the auspices of a medical aid program to a pediatrician at the age of 6.5 yr (M.R.) in Germany. He was born of healthy Iraqi parents after an uneventful pregnancy spontaneously at term. At the age of 2.5 yr, genital and scrotal hyperplasia was reported. The diagnosis of decreased cortisol secretion and elevated testosterone levels had been made at the University of Baghdad. At this time no therapy was started. At the chronological age of 4 yr, he showed an accelerated bone age of 8 yr, and treatment was initiated with glucocorticoids (drug and dose not available). One month before first admission in Germany, the glucocorticoid treatment was abandoned for unknown reasons. Initially he presented at age 6.6 yr with obesity, no hyperpigmentation, Tanner stages P1 and G3, and testicular volume of 1 ml each. ACTH was highly increased (494.35 pmol/liter; normal range 2.26–10.64 pmol/liter), 17-hydroxyprogesterone was moderately increased (8.47 nmol/liter; normal range 0.27–2.33 nmol/liter), and cortisol levels were decreased below the detection limit. The results of the multisteroid analysis (21) after a short ACTH test showed a typical pattern for 11OHD (Table 1Go).

Cases Z.P. and H.P. The patients were born of healthy Pakistani parents who are first-degree cousins. No adrenal disorders in the family history could be recollected. The male patient, Z.P., was born after an uneventful pregnancy in the 40th wk of gestation. His development was without pathological findings until the age of 4 yr, when he showed a growth acceleration. At the age of 5.1 yr, he was presented for the first time to a pediatric endocrinologist (J.C.) with bone age acceleration of 6 yr [bone age (BA) 11 yr], and Tanner pubertal stage P2, G3. No hypertension was found at this time. His sister, H.P., was born after an uneventful pregnancy in the 42nd wk of gestation. Retrospectively, she had a growth acceleration during the first years of life, growing at the 97th percentile. She was seen by a pediatric endocrinologist (J.C.) at the age of 3.6 yr, when a BA acceleration of 3.5 yr (BA 7 yr) was diagnosed. She had pubic hair Tanner stage P2 and a clitoral hyperplasia categorized as Prader genital stage II. Her blood pressure was mildly elevated (110/60 mm Hg). The measurement of multisteroids (21) revealed a typical pattern for 11OHD (Table 1Go). Plasma testosterone (Z.P.: 2.90 nmol/liter, normal range 0.07–0.22 nmol/liter; H.P.: 2.79 nmol/liter, normal range 0.07–0.22 nmol/liter) and androstenedione (Z.P.: 26.62 nmol/liter, normal range 0.77–1.82 nmol/liter; H.P.: 25.65 nmol/liter; normal range 0.24–0.73 nmol/liter) were also above the age- and gender-specific normal ranges in both patients.

Mutation analysis, construction of plasmids, and site-directed mutagenesis

Genomic DNA was prepared from peripheral blood leukocytes, using a standard protocol. The molecular genetic analysis was performed after PCR amplification by direct DNA sequencing the complete of the coding region of the CYP11B1 gene, including the intron-exon boundaries as described previously (12). The samples were electrophoresed on an automated ABI PRISM 310 sequencer and analyzed with the ABI SeqScape 1.1 software (PE Applied Biosystems, Foster City, CA).

The mutations were designated using the recommendations of the Nomenclature Working Group (22, 23), whereupon the prefix g indicates the genomic and c the cDNA sequence position.

The human full-length CYP11B1 cDNA cloned into the pSVL expression vector was kindly provided by Dr. Rita Bernhardt (Institute of Biochemistry, Universität des Saarlandes, Saarbrücken, Germany) (24). The mutagenesis was performed from the pSVL-CYP11B1 construct, using the QuikChange XL site-directed mutagenesis kit (Stratagene, Amsterdam, The Netherlands). The introduction of the W116C, L299P, and {Delta}F438 mutations was verified by sequencing the entire construct. To ensure the integrity of the expression plasmid, the mutated CYP11B1-cDNA fragments were recloned into the XbaI/BamHI site of a newly restricted pSVL expression vector, resulting in the pSVL-CYP11B1-W116C, pSVL-CYP11B1-L299P, and pSVL-CYP11B1-{Delta}F438 constructs.

In vitro expression and assays of enzyme activity

Approximately 2 x 106 COS-7 cells were transiently cotransfected using lipofectamine (Invitrogen, Karlsruhe, Germany) with 1 µg of each pSVL-CYP11B1 construct, 0.5 µg adrenodoxin, and 0.5 µg adrenodoxin reductase expression vectors pECE-Adx and pECE-AR, kindly provided by Dr. W. L. Miller (Department of Pediatrics, University of California, San Francisco, San Francisco, CA), and 1 µg ß-galactosidase pSV-Gal vector (Promega, Mannheim, Germany). Posttransfection treatment was performed following a standard protocol (Invitrogen) using DMEM supplemented with glutamine, antibiotics, and 10% fetal calf serum.

The 11-hydroxylase activity in intact COS-7 cells was measured 24 h after transfection. The cells were incubated for 24 h at 37 C with 1 ml full DMEM medium containing 0.2 µCi 3H-labeled 11-deoxycortisol and 250 nmol/liter unlabeled 11-deoxycortisol. After incubations, steroids were extracted from the culture medium with ice-cold methylene chloride, evaporated to dryness, and dissolved in ethanol. The steroids were separated by thin-layer chromatography (TLC) using methylene chloride-methanol-water (300:20:1, vol/vol/vol). The radioactivity was measured directly from the TLC plates using the Rita Star TLC-scanner (Ray Test, Straubenhardt, Germany) and analyzed using the version 1.97 software. Cells were trypsinized and lysed in reporter lysis buffer (Promega) followed by determination of protein content and measurement of ß-galactosidase activity according to the manufacturer’s protocol.

The 11-hydroxylase activity of the mutants was expressed as a percentage of substrate conversion in nanomoles per milligram–1 x minutes–1 taking the activity of cells expressing the CYP11B1 wild-type protein as 100% after correction for total protein and the activity of cells transfected with the empty pSVL plasmid. Calculation of enzymatic activities was performed using the GraphPad Prism software version 4.0 (GraphPad, Inc., San Diego, CA).

Western blot analysis was performed using an antihuman-CYP11B rabbit antiserum kindly provided by Dr. H. Takemori (Department of Molecular Physiological Chemistry, Osaka University Medical School, Osaka, Japan) in a standard protocol to ensure the expression and translation of the intact CYP11B1 wild-type and CYP11B1 mutant proteins.

Immunofluorescence

The immunofluorescence was performed using a standard protocol. The same antihuman-CYP11B rabbit antiserum used for the Western blot was used as primary antibody in combination with a mouse anti-Grp75 antibody (BIOMOL, Hamburg, Germany), as a mitochondrial marker, each in a 1:200 dilution. As secondary antibodies the antirabbit-ALEXA594 antibody (Molecular Probes, Leiden, The Netherlands) and the antimouse-fluorescein isothiocyanate antibody (Dianova, Hamburg, Germany) were used in 1:500 and 1:50 dilutions, respectively.

Molecular modeling

To substantiate the relatedness of the human CYP11B1 protein to the overall fold of the cytochrome P450 family revealed by homology analyses, we used a fold recognition algorithm to show that the human CYP11B1 sequence is compatible with the architecture of this family (ProHit package, ProCeryon Biosciences GmbH, Salzburg, Austria). The template structure with the highest score of the pair potential was the x-ray structure of the mammalian cytochrome CYP2C5 (PDB accession code 1DT6), which served as the template for the three-dimensional model of CYP11B1. According to the alignment obtained by the fold recognition procedure, amino acid residues were exchanged in the template. Insertions and deletions in CYP11B1 were modeled using a database-search approach included in the software package WHATIF (25). Finally, these model structures were energy minimized using the steepest descent algorithm implemented in the GROMOS-program package (van Gunsteren, W. F., BIOMOS biomolecular software b.v., Laboratory of Physical Chemistry, University of Groningen, The Netherlands). The structural representations were generated with the Ribbons program (26). All programs were run on a Silicon Graphics Indigo2 workstation (Silicon Graphics GmbH, Grasbrunn, Germany).


   Results
Top
 Abstract
 Introduction
 Patients and Methods
 Results
Discussion
 References
 
Mutation analysis

In all of the patients showing the hormonal pattern suggestive of an 11OHD (Table 1Go), mutations within the coding region of the CYP11B1 gene were identified either in homozygous or in compound heterozygous state, respectively.

Complete DNA sequencing of the CYP11B1 gene revealed a novel G to C point mutation at position bp 742 (corresponding to c.348G>C) in exon 2 of the CYP11B1 gene in homozygous state in patient A.T. and in heterozygous state in both parents (Fig. 1Go, A and B), confirming the segregation of this mutation. This mutation leads to the substitution of tryptophan to cysteine at amino acid position 116 (W116C) of the CYP11B1 protein.



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  		FIG. 1. Mutation analysis by direct DNA sequencing. A, The CYP11B1 gene and the localization of the novel mutations are depicted schematically. B, The base change from G to C at position bp 742 (corresponding to c.348G>C) leads to the substitution from tryptophan to cysteine at amino acid position 116. DNA sequencing of a healthy control shows the wild-type (WT) sequence, the parents (I.1 and I.2) are heterozygous, and the patient (II.1) is homozygous for the W116C mutation. C, The T at position bp 3522 is changed to C (corresponding to c.896T>C), leading to an amino acid substitution from leucine to proline at amino acid position 299 (L299P). The mutation was found in heterozygous state in the patient (II.1) and the father (I.1). D, The 3-bp deletion (TCT) at positions bp 4779–4781 (corresponding to c.1313_1315delTCT) leads to an in-frame deletion of phenylalanine at the amino acid residue 438 ({Delta}F438). The parents were found to be heterozygous for the deletion, whereas the mutation was detected in homozygous state in the two siblings (II.1, II.2) suffering from 11OHD.

 
Patient M.M. was found to be compound heterozygous for the as-yet-unknown T to C point mutation at bp 3522 (corresponding to c.896T>C) in exon 5 (paternal allele) (Fig. 1Go, A and C) and a previously described 1-bp deletion (g.4568delA; c.1180delA) in exon 7 (27). Since investigation of the maternal genotype was impossible because DNA material was unavailable, the segregation of the second mutation could not be confirmed. The g.3522T>C missense mutation results in a change from leucine to proline at amino acid position 299 of the 11-hydroxylase protein. The g.4568delA mutation results in a frameshift with predicted premature termination of a truncated protein (N394T... L429X).

The mutation analysis of patients Z.P. and H.P. elucidated a homozygous 3-bp deletion (g.4779–4781delTCT; c.1313_1315delTCT) in exon 8 of the CYP11B1 gene. This mutation was also found in heterozygous state in both parents and an unaffected sister (Fig. 1Go, A and C). The deletion is in-frame and results in the deletion of the phenylalanine at amino acid position 438 ({Delta}F438).

Because all of the described novel mutations are either missense or in-frame mutations, a further functional in vitro analysis was performed to clarify whether they were responsible for the phenotypical expression of CAH due to 11OHD in our patients.

Functional analysis of enzyme activity

The in vitro expression studies demonstrated that the W116C mutation reduced the 11-hydroxylase activity to 2.9 ± 0.9% (SD) for the conversion of 11-deoxycortisol to cortisol. Expression of the L299P mutant showed a reduction of enzymatic activity to 1.2 ± 0.9%. The {Delta}F438 mutation resulted in no measurable residual CYP11B1 activity (Fig. 2Go).



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  		FIG. 2. Residual 11-hydroxylase activity of the CYP11B1 mutants in transiently transfected intact COS-7 cells. The activity of the mutants is expressed in percent of wild-type (WT) activity, which is defined as 100%. Values are depicted for the conversion of 11-deoxycortisol to cortisol at a substrate concentration of 250 nmol/liter of unlabeled steroid (time of incubation, 24 h). The bars represent the mean ± 1 SD for a number (n) of determinations from different transfections.

 
The Western blot analysis of the wild-type and mutant proteins expressed in vitro demonstrated that none of the mutations affected the translation efficiency (data not shown). The expression and the correct intracellular localization of the wild type and mutants in the mitochondrial membrane were also shown by immunofluorescence studies of transiently transfected COS-7 cells (Fig. 3Go).



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  		FIG. 3. The left panel shows the mitochondrial localization of the human 11-hydroxylase by using antihuman-CYP11B rabbit polyclonal antiserum as primary and the antirabbit-ALEXA594 antibody as secondary antibody. In the middle panel, the mitochondrial Grp75 protein was marked by using a mouse anti-Grp75 antibody as primary and stained by the antimouse-fluorescein isothiocyanate antibody as secondary antibody. The intracellular colocalization of the CYP11B1 protein and the Grp75 protein in COS-7 cells is depicted in the right panel. WT, Wild type.

 

   Discussion
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
References
 
In the present study, we describe two novel missense mutations (W116C, L299P) and a novel in-frame deletion ({Delta}F438) within the CYP11B1 gene of patients suffering from classical 11OHD. Most of the CYP11B1-inactivating missense mutations described to date result in the classical form of 11OHD because the residual enzymatic activity of the mutants was found to be severely decreased or completely abolished (11, 12, 17, 19). These findings are consistent with the in vitro expression results of the three novel mutations described in this report.

Because the time span until diagnosis of CYP11B1 deficiency varied in our patients, it is not possible to assess whether these differences are due to phenotypical expression and disease severity or the inability to make a correct early diagnosis in these patients. It is well known that there is no good correlation between a specific mutation and the clinical phenotype in the classic form of 11OHD because there is a phenotypic variability in patients with the same CYP11B1 mutation regarding onset of disease and age of diagnosis, sexual ambiguity, and the expression of hypertension. However, no consistent correlation between the severity of hypertension and the degree of virilization could be detected either (5, 11, 20).

Our results clearly show that a residual 11-hydroxylase activity of 3% is insufficient to prevent from classic CAH due to CYP11B1 deficiency. This is also comparable with the phenotypical expression of classical CAH due 21OHD, which is caused by mutations of the CYP21 gene leading to a residual 21-hydroxylase activity of less than 5–10% of wild-type activity (1). The CYP11B1-inactiviating mutations P42S, N133H, and T319M were found to result in nonclassical 11OHD. These mutations reduced the in vitro enzymatic activity to 15–37% of wild-type activity (9). These results are also comparable with residual activities leading to the nonclassical form of 21OHD (1, 28, 29, 30). Intermediate forms between the simple virilizing and the nonclassic form of 21OHD have been characterized, caused by mutations with a residual enzyme activity between 5 and 15% of wild-type activity (31). Thus, we speculate that the same phenomenon might not yet be detected in 11OHD because to date no mutations have been found to result in residual activity within this intermediate activity range.

The structural model of the CYP11B1 protein based on the three-dimensional model of CYP11B1 built by using the x-ray structure of the mammalian CYP2C5 as a template (Fig. 4AGo) showed that the tryptophan 116 residue is localized in the B-C loop (Fig. 4Go, A and B). This part of cytochrome P450 enzymes contributes to substrate specificity because it contains the substrate recognition site 1, which is one of the six substrate recognition sites postulated in cytochrome P450 enzymes (32). The recently published structures of CYP2C5 (PDB code 1DT6) (33), CYP2C8 (PDB code 1PQ2) (34), and CYP2C9 (PDB code 1OG2) (35) illustrate that the B-C loop is located on the surface of the proteins and is not close to the heme (Fig. 4Go, A and B). It has been shown, that the B-C loop of the CYP2C9 protein plays a major role in substrate specificity (36, 37). Studying the structure of CYP2C5 revealed that the flexibility of the B-C loop is consistent with a conformational change required for substrate access when 4-methyl-N-methyl-N-(2-phenyl-2H-pyrazol-3-yl)benzenesulfonamide is bound (33). Further evidence has been provided for this region playing a critical role for specific catalytical activity of cytochrome P450 enzymes by substituting amino acid I112 in the human CYP11B2 protein (24). This study demonstrated the functional relevance of the amino acid residue 112 in the putative substrate access channel of CYP11B2. The W116 residue is conserved in the CYP11B1 and CYP11B2 enzymes of humans, mice (GenBank B41552; P15539), and rats (GenBank NP_036669; AAB60457, whereas it is not conserved in other cytochrome P450 enzymes (24). Moreover, a cluster of glycines is conserved in CYP11B1 and CYP11B2 (corresponding residues of human CYP11B1: G123, G128, G134), which are involved in the flexibility of the B-C loop. In particular, the side chains of W116 and L113 interact with F223 and W247 in a key-lock principle, anchoring the lid of CYP11B1 to the body of the molecule (Fig. 4BGo). Thus, the change from tryptophan to cysteine at amino acid position 116 (W116C) may influence the conformational change of the 11-hydroxylase protein necessary for substrate access and product release.



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  		FIG. 4. Three-dimensional molecular model of CYP11B1. N, Amino terminus; C, carboxy terminus. A, Total view of the three-dimensional model structure of CYP11B1. Black amino acid residues are depicted for W116, L299, and F438. The I helix is colored in brown, the B-C loop in light blue. B, The side chains of W116 and L113 interact with F223 and W247 in a key-lock principle, anchoring the lid of CYP11B1 to the body of the molecule. We hypothesize that the change from tryptophan to cysteine at amino acid position 116 (W116C) influences the conformational change of the 11-hydroxylase protein necessary for substrate access and product release. C, The interaction of the hydrophobic residues’ V290, L299, and I304 side chains is depicted. The amino acid I304 side chain is in contact with W137 neighboring residue R138, which interacts with one of the heme’s propionates. The introduction of a proline at residue 299 (L299P) of CYP11B1 changes the position of the I helix relative to the heme group and other parts of the CYP11B1 structure (in particular R138). Thus, we speculate that the severe decrease in in vitro 11-hydroxylase activity is the result of the steric disarrangement of the polar and apolar parts of the heme group relative to the enzyme.

 
The L299 residue is located N terminal from the putative I helix of the CYP11B1 protein (Fig. 4Go, A and C). The residue is highly conserved in human, mouse, and rat CYP11B1 and CYP11B2. The leucine residue is also found at the corresponding position of human steroid hydroxylases CYP21 (GenBank P08686), CYP17 (GenBank AAH63388, and CYP19A1 (GenBank P11511) and in human CYP4C1 (GenBank AAH17758. At least the hydrophobicity of this residue is also conserved in other human cytochrome P450 enzymes such as CYP2C9, CYP2C5, and CYP2C8, in which a phenylalanine is located at the corresponding position. The heme in cytochrome P450 enzymes is located between helices I and L. It has been shown by molecular modeling of CYP21 (38) and CYP17 (39) that the I helix plays a crucial role in substrate recognition and binding in steroid hydroxylases. Furthermore, it has also been demonstrated that helix I is part of the substrate binding pocket by solving the crystal structures of CYP2C9 (35), CYP2C5 (33), and CYP2C8 (34). The I helix of CYP11B1 and CYP11B2 also contains many hydrophobic amino acids and is the putative active site of CYP11B1 and CYP11B2 (40). Furthermore, it has been demonstrated by changing CYP11B2 to CYP11B1 corresponding residues and vice versa (41, 42) that the I helix of CYP11B1 and CYP11B2 is responsible for substrate specificity. The L299 side chain is in contact with the side chains of the hydrophobic residues V290 and I304. The I304 side chain is in contact with W137 neighboring residue R138, which interacts with one of the heme’s propionates. (Fig. 4CGo). Thus, the introduction of a proline at residue 299 (L299P) of CYP11B1 changes the position of the whole I helix relative to the heme group and other parts of the CYP11B1 structure involved in heme coordination. We speculate that the severe decrease in in vitro 11-hydroxylase activity is the result of a steric disarrangement of the polar and apolar parts of the heme group relative to the enzyme.

The {Delta}F438 mutation concerns a residue flanking a region involved in heme binding (24, 40) and is closely adjacent to the apolar part of the heme group in our three-dimensional model of CYP11B1 (Fig. 4Go). Hence, it can be hypothesized that either the steric arrangement of the heme group relative to the enzyme or the heme binding would be disturbed, resulting in the loss of 11-hydroxylase activity of the {Delta}F438 mutant.

In vitro expression analysis of CYP11B1 gene mutations in CAH patients due to 11OHD thus serves as a valuable tool for assessing the phenotypical expression and disease severity of 11OHD. Moreover, the combination of in vitro enzyme function and molecular modeling provides new insights in the understanding of cytochrome P450 structural-functional relationships.


   Acknowledgments
 
The authors are grateful to Dr. Rita Bernhardt for providing the CYP11B1 cDNA, Dr. Walter L. Miller for providing the Adx and AR cDNA, Wolfgang Sievert and Marc Rodinger for help with the molecular modeling, and Dr. Hiroshi Takemori for providing the antihuman-CYP11B rabbit antiserum. We appreciate the expert technical assistance of Gisela Hohmann and Brigitte Andresen and thank Joanna Voerste for linguistic help with the manuscript.


   Footnotes
 
First Published Online March 8, 2005

Abbreviations: BA, Bone age; CAH, congenital adrenal hyperplasia; 11OHD, 11-hydroxylase deficiency; 21OHD, 21-hydroxylase deficiency; TLC, thin-layer chromatography.

Received January 18, 2005.

Accepted March 2, 2005.


   References
Top
 Abstract
 Introduction
 Patients and Methods
 Results
 Discussion
 References
 

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