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The Journal of Clinical Endocrinology & Metabolism Vol. 85, No. 3 1261-1266
Copyright © 2000 by The Endocrine Society

Original Studies

Amino Acid Residue 147 of Human Aldosterone Synthase and 11ß-Hydroxylase Plays a Key Role in 11ß-Hydroxylation1

Angela Fisher2, Robert Fraser, John MC Connell and Eleanor Davies

Medical Research Council Blood Pressure Group, Department of Medicine and Therapeutics, Western Infirmary, Glasgow, United Kingdom G11 6NT

Address all correspondence and requests for reprints to: Dr. R. Fraser, Medical Research Council Blood Pressure Group, Department of Medicine and Therapeutics, Western Infirmary, Glasgow, United Kingdom G11 6NT. E-mail: rfraser{at}clinmed.gla.ac.uk


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
A number of amino acids differ between aldosterone synthase and 11ß-hydroxylase. To assess their importance in determining the different functional specificities, we substituted aldosterone synthase-specific (aspartate D147, isoleucine I248, glutamine Q43, and threonine T493) with 11ß-hydroxylase-specific amino acids (glutamate E147, threonine T248, arginine R43, and methionine M493), respectively. I248T, Q43R, and T493M had no effect on steroid production compared to wild-type aldosterone synthase. However, CYP11B2-D147E caused a significant increase in corticosterone production and a smaller increase in aldosterone production from 11-deoxycorticosterone (DOC). This appeared to be predominantly due to an increase in the 11ß-hydroxylation of DOC to corticosterone mediated by a decrease in Km, which was 1.4 µmol/L for the mutant compared with 5 µmol/L for the wild-type enzyme. CYP11B2-D147E had no effect on the conversion of 11-deoxycortisol to cortisol. The reverse construct (CYP11B1-E147D), substituting the 11ß-hydroxylase residue with the aldosterone synthase equivalent, decreased the conversion of DOC to corticosterone, which was mediated by an increase in Km that was 7.5 µmol/L for the mutant compared with 2.5 µmol/L for the wild-type enzyme. Again, the conversion of 11-deoxycortisol to cortisol was unimpaired. Thus, amino acid 147 is involved in the transformation of the 17-deoxysubstrate, but not the 17{alpha}-hydroxysubstrate. The results demonstrate that a conservative change in amino acid, even at some linear distance from known active centers, can significantly affect enzyme substrate affinity and subsequent steroid hormone production.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE GENES ENCODING 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2) lie in tandem on chromosome 8 in man and share 95% nucleotide homology (1, 2). Despite this, their products have quite different properties. 11ß-Hydroxylase is expressed in the zona fasciculata, is regulated by ACTH, and catalyses the conversion of 11-deoxycorticosterone (DOC), a weak mineralocorticoid, to corticosterone (B), 18-hydroxy-DOC, or 19-hydroxy-DOC. It also catalyzes the conversion of 11-deoxycortisol to cortisol, the principal glucocorticoid. Aldosterone synthase is expressed in the zona glomerulosa and is controlled by angiotensin II and potassium (3, 4, 5). It catalyses the initial 11ß-hydroxylation to convert DOC to B, but is then able to perform the necessary 18-hydroxylations and dehydration steps to transform this to 18-hydroxycorticosterone (18-OH-B) and then aldosterone, the principal mineralocorticoid. The contributions of several amino acids differing between the two enzymes to their different properties has been established (6, 7).

The relative efficiency of 11ß- and 18-hydroxylation will influence the ratio of glucocorticoid to mineralocorticoid secreted by the adrenal cortex. This is illustrated by the clinical effects of genetic abnormalities in the synthesis of these enzymes (8). Precise mutations for these are known (9, 10) that usually result in severe reduction or obliteration of activity together with abnormalities of blood pressure, electrolyte balance, and intermediary metabolism. However, more subtle changes may be implicated in essential and experimental hypertension. For example, abnormal steroid secretory patterns reported in essential hypertension are consistent with altered 11ß-hydroxylase activity, although the molecular basis for this has not been identified (11). Polymorphic differences in the CYP11B2 promoter are associated with both aldosterone secretion and blood pressure (12, 13). In the Dahl salt-resistant (SR) rat, mutations in the CYP11B1 and CYP11B2 genes result in decreased production of 18-OH-DOC and increased aldosterone production (14, 15, 16). The CYP11B1/2 locus is, therefore, an important candidate region in essential hypertension and cardiovascular disease, and recent reports suggest that this locus is one of the most highly polymorphic regions associated with essential hypertension (17).

In this study, amino acid residues differing between the two enzymes were studied by site-directed mutagenesis and subsequent biosynthetic and kinetic analysis. Two of the residues are from exons 3 and 4, a region of the gene already known to be important in determining the steroid intermediate phenotype of the Dahl rat.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Plasmids

The expression plasmids pCMV4-B1 and pCMV4-B2 containing the complementary DNA for human 11ß-hydroxylase (CYP11B1) and aldosterone synthase (CYP11B2), respectively, were provided by Prof. Perrin C. White (University of Texas Southwestern Medical Center, Dallas, TX) (18). pCD-adrenodoxin (19) was provided by Prof. J. Ian Mason (Department of Clinical Biochemistry, University of Edinburgh, Edinburgh, UK). pSV-ß-gal was used as a reporter gene (Promega Corp., Madison, WI).

Site-directed mutagenesis

Site-directed mutagenesis was performed using the Quick-Change site-directed mutagenesis kit (Stratagene, Cambridge, UK). Mutation of nucleotides encoding amino acids aspartate 147, isoleucine 248, glutamine 43, and threonine 493 of aldosterone synthase to the 11ß-hydroxylase equivalents (glutamate 147, threonine 248, arginine 43, and methionine 493) was carried out by PCR, using sense and antisense primers incorporating the desired mutation (Table 1Go; Oswel DNA Service, University of Southampton, Southampton, UK). The converse mutation of glutamate 147 of 11ß-hydroxylase to aspartate, the aldosterone synthase equivalent, was also carried out. The PCR reaction contained 5 µL reaction buffer (10x), 2 µL plasmid DNA (pCMV4-B2, 20 ng/µL), 1 µL (125 ng) each of sense and antisense primer, 1 µL deoxy-NTP mix (10 mmol/L), 1 µL Pfu DNA polymerase (2.5 U/µL), and 40 µL H2O. The PCR procedure consisted of an initial denaturation step at 95 C for 30 s, followed by 12 cycles of denaturation at 95 C for 30 s, annealing at 55 C for 1 min, and extension at 68 C for 14 min. The parental (nonmutated) supercoiled dsDNA template, unlike the mutated strand, is methylated and can therefore be digested with the methylation-sensitive restriction enzyme DpnI for 1 h at 37 C. Epicurion Coli XL1-Blue supercompetent cells (50 µL; Stratagene, Cambridge, UK) were transformed with 1 µL PCR product by heat shock at 42 C for 45 s and grown at 37 C in NZY+ broth for 1 h. The transformation reaction medium was plated onto Luria Bertoni/ampicillin plates (100 µg/mL) and incubated at 37 C for more than 16 h. Plasmid DNA was prepared, and the entire insert was sequenced to ensure that the desired mutation had been incorporated. Large scale plasmid DNA preparations were carried out by CsCl centrifugation.


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  		Table 1. Oligonucleotides used for site-directed mutagenesis and sequencing

 
COS cell transfection

COS-7 cells (European Cell Culture Collection, Salisbury, UK) were maintained at 37 C in 5% CO2 in DMEM supplemented with 5% newborn calf serum, 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.025 mg amphotericin B (antibiotic-antimycotic solution, Sigma-Aldrich Corp., Poole, UK). Transfections were carried out using N-[1-(2,3-Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate liposomal transfection reagent (Roche Molecular Biochemicals, Mannheim, Germany). Approximately 5 x 106 COS-7 cells at 80% confluence were preincubated for 3 h in 10 mL Optimem I reduced serum medium supplemented with 100 U/mL penicillin, 0.1 mg/mL streptomycin, and 0.025 mg amphotericin B (antibiotic-antimycotic solution, Sigma-Aldrich Corp.). A transfection mixture prepared in 20 mmol/L HEPES buffer (Sigma-Aldrich Corp.) containing 10 µg pSV-ß-galac-tosidase, 5 µg pCD-Adx, 10 µg test plasmid, and 75 µL N-[1-(2,3- Dioleoyloxy)propyl]-N,N,N-trimethylammonium methylsulfate was added to the medium and left for 8 h. This was subsequently removed, and the cells were incubated for 24 h with supplemented DMEM as described above. The test plasmid was wild-type CYP11B1, wild-type CYP11B2, CYP11B2-Q43R, CYP11B2-D147E, CYP11B1-E147D, CYP11B2-I248T, or CYP11B2-T493M. Control transfections were also performed.

Initial steroid conversion experiments

After 24 h, the cells were permeabilized with 10% dimethylsulfoxide and incubated with DMEM supplemented with enzyme substrate. In these initial experiments, 5 µmol/L DOC plus [3H]DOC (10,000 cpm; 36 Ci/mmol) or 5 µmol/L 11-deoxycortisol plus [3H]11-deoxycortisol (10,000 cpm; 36 Ci/mmol) were incubated with the cells for 48 h. The medium was retained for steroid analysis, and cell extracts were prepared for protein measurement and determination of ß-galactosidase activity. The conversions of [3H]DOC to B, 18-OH-B, and aldosterone and of [3H]11-deoxycortisol to cortisol were measured. Steroids were extracted from 1-mL aliquots of medium with freshly distilled methylene chloride. The organic phase was washed with dH2O (1 mL), and unlabeled DOC, B, 18-OH-B, and aldosterone (3 µg) were added as carrier. The extract was evaporated to dryness under a stream of N2 at 37 C, and the residue was chromatographed on glass-backed silica F254-coated TLC plates. The plates were developed in methylene chloride-methanol-water (300:20:1). Steroids were located under UV light, identified by concurrently chromatographed standards (3 µg), and eluted in ethanol. The 3H content of the eluates were measured by liquid scintillation spectrometry.

Steroid production rate and kinetic experiments

In this second set of experiments, the cells transfected with wild-type CYP11B1 or CYP11B2 and mutant CYP11B1-E147D or CYP11B2-D147E were incubated for 8 h with various concentrations of DOC (0.01–10 µmol/L). Preliminary time-course experiments were performed using the B production rate to determine the time point at which steroid production was nearing the maximum, but was still within the linear range that follows first rate order kinetics. An end point of 8 h was therefore chosen, and the culture medium was retained for steroid RIA (20). Cell extracts were prepared for protein and ß-galactosidase activity measurements.

Protein and ß-galactosidase measurements

COS-7 cell extracts were prepared after transfection for measurements of protein content (Bio-Rad Laboratories, Inc., Hemel Hempstead, UK) and ß-galactosidase activity (Promega Corp. UK Ltd., Southhampton, UK) following the manufacturer’s protocols. Transfection efficiency was determined using these data, and steroid results were corrected appropriately. The initial steroid conversion ratio experiments were analyzed by Mann-Whitney U test, and the second set of experiments measuring steroid production rates were analyzed by ANOVA and Student’s t test where appropriate.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Steroid conversion ratios

After 48 h of treatment with substrate, CYP11B2-D147E significantly increased the conversion of DOC to B from 0.53 ± 0.05 to 3.05 ± 0.37 (P < 0.001, by U test) compared to the wild-type aldosterone synthase, suggesting that 11ß-hydroxylase function was increased (Fig. 1Go). CYP11B2-D147E also caused a small, but nonetheless significant, decrease in the 18-OH-B to B ratio from 0.98 ± 0.08 to 0.68 ± 0.09 (P < 0.05, by U test). There was no significant difference in the ratio of aldosterone to 18-OH-B or aldosterone to B between the wild-type aldosterone synthase and CYP11B2-D147E. CYP11B2-I248T had no effect on any of the ratios measured, nor did CYP11B2-Q43R or CYP11B2-T493M (data not shown). As 11ß-hydroxylation efficiency was increased, the conversion of DOC to B by CYP11B2-D147E was compared with that by wild-type 11ß-hydroxylase (Fig. 1Go). The B to DOC ratio for the wild-type 11ß-hydroxylase (6.2 ± 0.41) was high compared to that for wild-type aldosterone synthase (0.53 ± 0.05; P < 0.001). The B to DOC ratio for CYP11B2-D147E was intermediate (3.05 ± 0.37). Wild-type aldosterone synthase had minimal ability to convert 11-deoxycortisol to cortisol, and this was not altered by either CYP11B2-D147E or the CYP11B2-I248T mutant (data not shown). Therefore, further kinetic analysis was not carried out.



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  		Figure 1. Effects of mutants CYP11B2-D147E and CYP11B2-I248T compared to wild-type aldosterone synthase and 11ß-hydroxylase on steroid ratio (product to substrate). Transfected COS-7 cells were incubated with 5 µmol/L DOC for 48 h. Values are the mean ± SEM (n = 8).

 
The reverse mutant was also studied. 11ß-Hydroxylation by CYP11B1-E147D was significantly decreased compared to that of wild-type 11ß-hydroxylase (DOC to B ratio, 9.45 ± 2.66 vs. 2.11 ± 1.28; P < 0.0001, by U test; Fig. 2Go). Both were more efficient than the wild-type aldosterone synthase (DOC to B ratio, 0.63 ± 0.08; P < 0.001, by U test). This suggests that the CYP11B1-E147D mutant decreased 11ß-hydroxylase efficiency compared to wild-type 11ß-hydroxylase, but that it was still more efficient than aldosterone synthase. The CYP11B1-E147D mutation had no effect on the efficiency of 11ß-hydroxylation of 11-deoxycortisol to cortisol (data not shown). Therefore, further kinetic analysis was not carried out.



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  		Figure 2. Effect of mutant CYP11B1-E147D compared to wild-type aldosterone synthase and 11ß-hydroxylase on the B to DOC ratio. Transfected COS-7 cells were incubated with 5 µmol/L DOC for 48 h. Values are the mean ± SEM (n = 8).

 
Steroid production rates and kinetic analysis

Aldosterone synthase mutant CYP11B2-D147E. Dose-response curves of the steroid products, corticosterone, 18-OH-B, and aldosterone after 8 h of treatment with various substrate (DOC) concentrations are shown in Fig. 3Go, a–c. B, 18-OH-B, and aldosterone production by CYP11B2-D147E was significantly increased at all concentrations of DOC compared to the effect of wild-type aldosterone synthase. Kinetic analysis revealed that mutant CYP11B2-D147E has increased affinity for DOC compared to wild-type aldosterone synthase. The apparent Km values of wild-type aldosterone synthase and the mutant CYP11B2-D147E for the conversion of DOC to B were 5 and 1.4 µmol/L, respectively (P < 0.00005, by ANOVA; Lineweaver-Burke analysis not shown). The maximum velocity was not measured, as it requires an accurate measurement of enzyme concentration. Quantitation by SDS-PAGE immunoblotting is impossible, because currently there are no specific antibodies against human 11ß-hydroxylase and aldosterone synthase.



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  		Figure 3. a–c, Effect of mutant CYP11B2-D147E compared to wild-type aldosterone synthase on the conversion of DOC (0.01–10 µmol/L) to B, 18-OHB, and aldosterone. Transfected COS-7 cells were incubated with the various concentrations of DOC for 8 h. Values are the mean ± SEM (n = 4).

 
11ß-Hydroxylase mutant CYP11B1-E147D. Dose-response curves of steroid products B and 18-OH-B against substrate concentration for 11ß-hydroxylase and the mutant CYP11B1-E147D are shown in Fig. 4Go, a and b, respectively. B production by CYP11B1-E147D was significantly decreased at all substrate concentrations compared to the effect of wild-type 11ß-hydroxylase. The apparent Km values of wild-type 11ß-hydroxylase and the mutant CYP11B1-E147D for the conversion of DOC to B were 2.5 and 7.5 µmol/L, respectively (P < 0.00005, by ANOVA; Lineweaver-Burke analysis not shown). 18-OHB production by CYP11B1-E147D was also decreased compared to that by wild-type 11ß-hydroxylase (P < 0.001, by ANOVA), but to a lesser extent than B production. Apparent Km values were not measured.



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  		Figure 4. a and b, Effect of mutant CYP11B1-E147D compared to wild-type 11ß-hydroxylase on the conversion of DOC (0.5–10 µmol/L) to B and 18-OHB. Transfected COS-7 cells were incubated with the various concentrations of DOC for 8 h. Values are the mean ± SEM (n = 4).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The contrasting catalytic potential of human 11ß-hydroxylase and aldosterone synthase must be due to the small differences in their amino acid sequences. Molecular studies by other groups have already shown the absolute requirement of a glycine residue at position 288 and an alanine residue at position 320 of aldosterone synthase for efficient 18-hydroxylation and subsequent 18-oxidation (7). Although the precise three-dimensional structure of mammalian mitochondrial P-450 enzymes is unknown, comparison with their bacterial homologs places these residues in the key substrate-binding and catalytic domains (7). However, residues adjacent to or even more distant from this site may also modify enzyme activity. In selecting which residues to study, we were guided in part by the Dahl salt-resistant (SR) and salt-sensitive (SS) rats, which have been used to investigate the genetic component of salt-sensitive hypertension. In the SS strain, there is increased 18-hydroxy-DOC production, which accounts for approximately 20% of the excess blood pressure in this strain. Moreover, the SR strain has mutations of CYP11B1 that decrease 18-hydroxy-DOC production relative to that of corticosterone and cosegregate with resistance to the hypertensinogenic effects of salt (14, 15, 16). Studies of the CYP11B2 gene have identified seven mutations in the Dahl SR rat, two of which alter the predicted amino acid composition of the protein (exon 3, Glu136 to Asp; exon 4, Gln251 to Arg). These encode an enzyme with a greater apparent maximum velocity and a lower apparent Km, resulting in an increased rate of conversion of DOC to aldosterone (15). Replication of these mutations in human aldosterone synthase increased B and aldosterone production (21). Examination of exons 3 and 4 of the human genes shows six codons that differ between CYP11B1 and CYP11B2, four of which code for different amino acids. The role of two of these amino acids in conferring functional specificity of the enzymes has been examined in this study (amino acids 147 and 248). In addition, two other amino acids, which differ between the two enzymes but lie distal to the putative active site, have been studied (amino acids 43 and 493). We substituted aldosterone synthase-specific amino acids (aspartate D147, isoleucine I248, glutamine Q43, and threonine T493) with 11ß-hydroxylase-specific amino acids (glutamate E147, threonine T248, arginine R43, and methionine M493). Alteration of residues 248, 43, and 493 of aldosterone synthase had no effect on enzyme function, suggesting that these loci do not contribute to the functional specificity of the enzyme. A similar conclusion was reached by Fardella et al. for residue 248 (22). In contrast, substituting the aspartate 147 residue of aldosterone synthase for the corresponding 11ß-hydroxylase-specific glutamate residue caused a marked increase in B production and a smaller increase in aldosterone production. Closer inspection of the constituent reactions of aldosterone synthase revealed that there was a large increase in the ratio of B to DOC and a small decrease in the 18-OH-B to B ratio, whereas the aldosterone to 18-OH-B and aldosterone to B ratios were unaffected. This suggests that the increase in B and aldosterone production is due to an increase in the 11ß-hydroxylase activity of aldosterone synthase and not to increased 18-hydroxylase/oxidase activity. This would not have been apparent from the work of Fardella et al. (22), who studied this residue but measured only the end product, aldosterone. Subsequent kinetic analysis showed that mutation of residue 147 increased the affinity of aldosterone synthase for DOC, as shown by a decrease in the apparent Km of the enzyme. However, despite the enhancement, activity remained below that of wild-type 11ß-hydroxylase.

Conversely, mutating 11ß-hydroxylase at residue 147 to the aldosterone synthase equivalent significantly reduced the 11ß-hydroxylation capacity, although the activity still remained higher than that of wild-type aldosterone synthase. Kinetic analysis revealed that mutation of residue 147 of 11ß-hydroxylase decreased the affinity of the enzyme for DOC, as shown by an increase in the apparent Km of the enzyme. Taken together, these results clearly implicate residue 147 in contributing to the efficiency of 11ß-hydroxylation, but suggest that other residues are also involved. Indeed, studies have shown that amino acid residues 301, 302, 320, and 384 are also involved in 11ß-hydroxylation (6). In the initial studies, Western analysis was not carried out due to the lack of suitable specific antibodies for human aldosterone synthase and 11ß-hydroxylase, and it could be argued that the changes in steroid production observed may be due to differences in the expression of the wild-type and mutated enzymes, although this seems unlikely, as only selected functions were affected. However, the Km is a measurement that is independent of enzyme concentration, and the change observed here reflects true differences in enzymatic activity.

The use of B and 18-OH-B as substrates would provide a clearer picture of the effects of CYP11B2-D147E on 18-modifying functions of the enzyme, but these make poor substrates for aldosterone synthase (23), and the affinities of the enzymes for these substrates were not determined by ourselves or others (6, 7). Although mutant CYP11B2-D147E enhanced the ability of aldosterone synthase to convert DOC to B, it did not change the conversion of 11-deoxycortisol to cortisol. Moreover, the CYP11B1-E147D mutant also created an 11ß-hydroxylase with unimpaired ability to synthesize cortisol. Therefore, the function of residue 147 appears to be substrate dependant, and it is possible that the 17{alpha}-hydroxyl group of 11-deoxycortisol interacts with the enzyme in a way that obviates the role of this amino acid.

The effect of substituting amino acid residue 147 from 11ß-hydroxylase to aldosterone synthase and vice versa on 11-hydroxylase activity is surprising, in that the amino acid change is conservative (acidic for acidic). Studies of bacterial homologues suggest that P-450 enzymes consist of a number of helices (A to L) (24). Residue 147 is probably situated at the interface between the D helix and a stretch of rope, and it may interact in some way with the substrate and heme-binding domains, possibly to maintain the correct orientation of the substrate within the active site.

Naturally occurring gene rearrangements at the CYP11B1/ B2 locus are well characterized. In glucocorticoid-remediable hyperaldosteronism, the proximal exons of CYP11B1 and the distal exons of CYP11B2 form a chimera as a result of unequal recombination at meiosis (25). The cross-over site varies between kindreds (26). Indeed, some studies have shown that glucocorticoid-remediable hyperaldosteronism patients may have reduced 11ß-hydroxylase activity (27). Although Pascoe et al. (28) studied the effect of serial exon interchanges between CYP11B2 and CYP11B1, 11ß-hydroxylase function was not measured. It is possible that the variation in this activity might depend on the source of residue 147.

Thus, a conservative change in the amino acid composition of these enzymes can significantly change steroid production, although as yet there is no published evidence to suggest that alterations in codon 147 are present in a normotensive or a hypertensive population. However, other polymorphisms in these genes have been identified, e.g. residue 173 of aldosterone synthase in a group of subjects with low renin essential hypertension; this, however, had no effect on enzyme kinetics in vitro (29). Our own screening studies have also identified sites within the enzymes where the 11ß-hydroxylase and aldosterone synthase-specific residues are transposed (unpublished data), and these may have functional implications. Although it is tempting to ignore the potential effects of these and other polymorphisms coding for amino acid changes that are conservative or distant from known active centers, this study shows that further evaluation of their biochemical and clinical phenotypes may be rewarding.


   Acknowledgments
 
We thank Dr. Rita Bernhardt (Saarland University, Saarbrucken, Germany) and Dr. C. Gomez Sanchez (University of Missouri, Columbia, MO) for valuable advice.


   Footnotes
 
1 This work was supported by Medical Research Council Program Grant G9317119 and a West Glasgow National Health Service Trust Research Grant. Back

2 Current address: Organon Laboratories, Newhouse, Lanarkshire, United Kingdom ML1 5SH. Back

Received August 25, 1999.

Revised November 19, 1999.

Accepted December 9, 1999.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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