Molecular and Cellular Endocrinology 215 (2004) 149–159

Review
Modulation of aldosterone and cortisol synthesis on the molecular level
Michael Lisurek, Rita Bernhardt∗
Universität des Saarlandes, FR 8.8 Biochemie, Postfach 151150, 66041 Saarbrücken, Germany

Abstract

CYP11B1 and the closely related CYP11B2 are involved in the production of adrenal steroid hormones. Although in human their primary
structure is 93% identical they are involved in the biosynthesis of functionally diverse products, such as glucocorticoids and mineralocorticoids,
respectively. In contrast, bovine CYP11B1 combines both activities in one single enzyme. The CYP11B family belongs to class I cytochromes
P450 that have been described in bacteria and mitochondria and receive their electrons from a low molecular weight iron sulphur protein
which is reduced by a NADPH-dependent FAD-containing reductase.
In this review, we summarise the current knowledge on the modulation of aldosterone and cortisol synthesis by transcriptional regulation,
on the molecular level as consequence of mutations found in patients suffering from steroid hormone-related diseases as well as introduced by
site-directed mutagenesis and as consequence of protein–protein interaction with both CYP11A1 and the natural redox partner adrenodoxin.
© 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: CYP11B1; CYP11B2; Cytochrome P450; 11␤-Hydroxylase; Aldosterone synthase; Glucocorticoids; Mineralocorticoids

1. Introduction strated that hyperaldosteronism may be accompanied by

The most common reaction catalysed by cytochromes
P450 is hydroxylation of organic compounds, but they are
also capable of performing a wide spectrum of different re-
actions including N-oxidation, N-, S-, and O-dealkylation,
sulfoxidation, epoxidation, peroxidation, deamination,
desulfuration, dehalogenation and N-oxide reduction (Ortiz
De Montellano, 1995; Bernhardt, 1996). Cytochromes P450
are key enzymes responsible for the metabolism of drugs
and other xenobiotics and the anabolism of important en-
dogenous compounds like steroid hormones, bile acids or
prostaglandins.
In humans the final steps of cortisol and aldosterone
synthesis are performed by 11␤-hydroxylase (CYP11B1)
and aldosterone synthase (CYP11B2), respectively (Mornet
et al., 1989; Kawamoto et al., 1990a,b). Cortisol is the main
glucocorticoid in humans. It regulates energy mobilisation,
stress response and is additionally involved in the immune
response of the human body. Aldosterone is the most po-
tent natural human mineralocorticoid. It participates in the
regulation of the salt and water household of the body and
thus in the regulation of blood pressure. It has been demon-
∗ Corresponding author. Tel.: +49-681-302-4241;
fax: +49-681-302-4739.
E-mail address: ritabern@rz.uni-sb.de (R. Bernhardt).
hypertension and heart disease (see below).
CYP11B enzymes of other species have also been stud-
ied, and it could be shown that also in the adrenal cor-
tex of rat (Matsukawa et al., 1990), mouse (Domalik et al.,
1991), guinea pig (Bülow et al., 1996; Bülow and Bernhardt,
2002), baboon (Hampf et al., 1996; Swart et al., 2000)
and hamster (Veronneau et al., 1996), two distinct isoforms
are involved in the formation of either mineralo- or glu-
cocorticoids. In contrast, in cows (Wada et al., 1985), pigs
(Yanagibashi et al., 1986), sheep (Boon et al., 1995) and
frogs (Nonaka et al., 1995) the synthesis of gluco- and
mineralocorticoids is catalysed by a single enzyme, termed
CYP11B1. In rat a third CYP11B isoform is expressed
with 11␤- and 18-hydroxylase activities, but no detectable
18-oxidase activity (Mellon et al., 1995).
2. Aldosterone biosynthesis
Human CYP11B1 and CYP11B2 catalyse the final steps
in gluco- and mineralocorticoid synthesis, respectively
(Fig. 1). CYP11B1 is expressed at high levels in the zona
fasciculata/reticularis of the adrenal cortex (Erdmann et al.,
1995a,b) and produces cortisol, the principal human glu-
cocorticoid. In contrast, aldosterone secretion (Miller and
Tyrell, 1995) and CYP11B2 expression (Pascoe et al., 1995)

0303-7207/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.mce.2003.11.008
150 M. Lisurek, R. Bernhardt / Molecular and Cellular Endocrinology 215 (2004) 149–159
Fig. 1. Reactions catalysed by human CYP11B1 and CYP11B2: (A) CYP11B1 catalyses the 11␤-hydroxylation of 11-deoxycortisol to cortisol; (B)
CYP11B2 converts 11-deoxycorticosterone via corticosterone and 18-hydroxycorticosterone to aldosterone.
take place at low levels in the zona glomerulosa of the
adrenal cortex. This zonal distribution of expression has
recently been further investigated with surgically removed
human adrenal gland cells, showing a higher concentration
of CYP11B1 in the zona fasciculata than in the zona reticu-
laris (Young et al., 2003). Cortisol is usually secreted 100- to
1000-fold in excess over aldosterone, but this is not only due
to transcriptional regulation of these enzymes, but also to dif-
ferences in catalytic activities. The adrenal steroid hormones
are produced in multi-step pathways involving five differ-
ent cytochromes P450 and 3␤-HSD, a member of the short
chain dehydrogenase family (Fig. 2): CYP11A1 (side chain
cleavage enzyme), CYP17 (steroid 17␣-hydroxylase and
17,20-lyase), CYP21 (steroid 21-hydroxylase), CYP11B1
(steroid 11␤-hydroxylase) and CYP11B2 (aldosterone syn-
thase) (Bernhardt, 1996; Bureik et al., 2002a). In vitro
reconstitution of the CYP11B1 electron transfer chain
showed that 11-deoxycorticosterone and, to a minor extent,
11-deoxycortisol are 11␤-hydroxylated (Kawamoto et al.,
1990a; Curnow et al., 1991; Denner et al., 1995; Fisher
et al., 2001). In contrast to this, human CYP11B2 exhibits
a weak 11␤-hydroxylase activity towards 11-deoxycortisol,
but 11␤-hydroxylates 11-deoxycorticosterone to corticos-
terone and in addition, 18-hydroxylates and 18-oxidises
it further to 18-hydroxycorticosterone and aldosterone,
respectively (Fig. 1). CYP11B2 can also 18-hydroxylate
and 18-oxidise cortisol. Bovine CYP11B1, producing both
gluco- and mineralocorticoids, can be purified from adreno-
cortical mitochondria (Akhrem et al., 1979). Kinetic studies
have shown that the two intermediates leading to aldos-
terone from 11-deoxycorticosterone are not released from
the active site of the enzyme (Imai et al., 1998). This can
also be expected to be the case for human CYP11B2, since
it is known that the intermediates are poor substrates for
human CYP11B2 (Denner et al., 1995).
Both CYP11B1 and CYP11B2 expression have also been
detected in extra adrenal tissue such as rat brain (Ozaki et al.,
1991; Erdmann et al., 1996; MacKenzie et al., 2000) and
heart (Rudolph et al., 2000; Yoshimura et al., 2002). Even
if their function in most of these tissue is not fully under-
stood, it is known that chronic overproduction of aldosterone
leads to an increased blood volume and stimulates cardiac
hypertrophy, myocardial fibrosis and ventricular arrhythmia
(Brilla, 2000; Lijnen and Petrov, 2000). In the heart, further
pathological consequences such as endothelial dysfunction
and tissue damage are observed by aldosterone dysregula-
tion (Takeda et al., 2000).
3. Differences and similarities between CYP11B1 and
CYP11B2
Human CYP11B1 and CYP11B2 are both synthesised
as 503 amino acids containing precursor proteins (Mornet
et al., 1989; Kawamoto et al., 1990a), both containing a
24-residue N-terminal mitochondrial targeting sequence
which is cleaved off after translocation into the mitochon-
drial matrix. In the mature enzymes, only 29 out of 479
 M. Lisurek, R. Bernhardt / Molecular and Cellular Endocrinology 215 (2004) 149–159
Fig. 2. Steroid biosynthesis in the adrenal cortex. The diagram shows the reactions leading from cholesterol to cortisol, aldosterone and androstenedione
including the names of the corresponding enzymes and the organelles in which the reactions are carried out.
residues are not identical (Belkina et al., 2001). All of
these residues seem to be located outside of the generally
accepted substrate recognition sites (Gotoh, 1992) and are
spread throughout the protein (Fig. 3). Human CYP11B1
Fig. 3. Distribution of the amino acids differing between human CYP11B1
and CYP11B2. The C␣-positions of the exchanged amino acid are labelled
and shown as black balls on the ribbon structure of the homology model
of CYP11B2 (Belkina et al., 2001). The heme is represented as grey stick
model. Amino acids 301, 302 and 320 can be found in the I-helix.
151
and CYP11B2 have apparent molecular masses of 50 and
48.5 kDa, respectively, and are bound to the inner mito-
chondrial membrane by as yet undefined protein segments
(Ogishima et al., 1991).
Understanding the structure–function relationships
of CYP11B enzymes requires information about their
three-dimensional structure. Protein structure determina-
tion by X-ray diffraction is often problematic in case of
membrane-bound proteins such as CYP11B1 and CYP11B2,
and NMR structure determination is restricted to smaller
proteins. Due to these reasons, no structure of a mitochon-
drial cytochrome P450 has been experimentally resolved so
far. Until recently only the structures of several bacterial
cytochromes P450 and that of a single microsomal P450
(CYP2C5), solubilised by truncation and site-directed mu-
tagenesis (Cosme and Johnson, 2000) has been determined
experimentally (Williams et al., 2000). While this paper was
in preparation, one more structure of a membrane bound
cytochromes P450 (CYP2C9) became available (Williams
et al., 2003). Analysis of the structures revealed a conserved
structural fold. Therefore, homology modelling studies of
human CYP11B1 and CYP11B2 have been performed. The
models have been evaluated and used to explain the signif-
icance of a number of residues that were identified either
by mutagenesis studies or found in patients (Belkina et al.,
2001).
152 M. Lisurek, R. Bernhardt / Molecular and Cellular Endocrinology 215 (2004) 149–159
Fig. 4. Superposition of the ribbon structures of the homology models
of human CYP11B1 (black) and CYP11B2 (grey) (Belkina et al., 2001).
Helices are labelled in the letter code. While the overall structure is similar,
the position of the hemes in both proteins is different. The orientation of
the model is essentially as shown in Fig. 3.
These models suggest that the main difference between
the two proteins is the position of the heme. An angle of
approximately 20◦ between the hemes of the two models
has been observed, apparently dependent on the interaction
of side-chains forming the heme environment and the ori-
entation of its binding loop (Fig. 4). In case of CYP11B1,
one heme propionate group forms a hydrogen bond with
Arg448 while the second interacts with Arg384, whereas
in CYP11B2 both heme propionate groups are involved
in hydrogen bond interactions with Arg448. Both, Arg384
and Arg448, have been found to be mutated in CYP11B1
of patients suffering from congenital adrenal hyperplasia
(CAH) (Curnow et al., 1993; Nakagawa et al., 1995; Geley
et al., 1996; White et al., 1991) (see below). All known
mutations in positions 384 and 448 led to a complete loss
of enzyme activity, most probably due to destabilisation of
the holoprotein. As a consequence of the different hydrogen
bonding network around Arg384, Arg448 and the heme
propionates, the active site of CYP11B2 is predicted to be
smaller than that of CYP11B1. The larger active site found
in the CYP11B1 model correlates with the fact that the
natural substrate of CYP11B1 (11-deoxycortisol) is larger
than that of CYP11B2 (11-deoxycorticosterone) due to the
presence of an additional 17␣-hydroxy group (Fig. 1).
4. Regulation on the level of transcription
The coding sequences of the CYP11B1 and CYP11B2
genes are highly homologous, whereas their promoter re-
gions are strikingly different, emphasising the fact that they
are regulated differently (Lifton et al., 2001; White, 2001;
Ferrari, 2002). Usually CYP11B1 activity is regulated by
the adrenocorticotropic hormone (ACTH) (Waterman and
Simson, 1989), and the expression of the CYP11B2 gene
is mainly controled by the renin–angiotensin system (Quinn
and Williams, 1988). Angiotensin II and potassium increase
the production levels of CYP11B2, but aldosterone synthe-
sis is also influenced by other factors such as sodium status
or the atrial natriuretic peptide (ANP).
A -344 C/T polymorphism in the promoter region of hu-
man CYP11B2 has been associated with transcriptional reg-
ulation of this gene (Kupari et al., 1998; Lim et al., 2002),
but so far experimental evidence have not underlined this as-
sumption leading to a controversial discussion of the matter
(Schunkert et al., 1999; Hautanen et al., 1999; Delles et al.,
2001; Connell et al., 2003).
The CYP11B1 and CYP11B2 genes are arranged
tandemly on chromosome 8, approximately 40 kb apart
from each other (Chua et al., 1987; Wagner et al., 1991).
Recent studies have mapped human CYP11B2 to the cy-
togenic band 8q24.3 (Taymans et al., 1998). Chimeric
enzymes of CYP11B1 and CYP11B2, derived by unequal
crossing over, lead to severe consequences as displayed in
patients suffering from familial hyperaldosteronism type I
(also called glucocorticoid remediable hyperaldosteronism)
(Lifton et al., 1992; MacConnachie et al., 1998; Seeman
et al., 1999). The chimeric genes are composed of the
5 -coding sequence and regulatory elements of CYP11B1
fused to the 3 -coding sequence of CYP11B2 and exhibit
aldosterone synthesis under the regulatory control of the
ACTH-sensitive promoter, thus leading to hypersecretion of
aldosterone. Administration of dexamethasone (or any other
glucocorticoid) suppresses ACTH production and results in
corrections of the biochemical anomaly. Patients suffering
from familial hyperaldosteronism type I carrying genes
consisting of the first four exons of CYP11B1 and the last
five exons of CYP11B2 have been found. Transfection ex-
periments with chimeras, where only the last four exons of
CYP11B1 were replaced by the corresponding ones found
in CYP11B2, showed that these constructs had activities
similar to cells transfected with CYP11B1 and no aldos-
terone formation could be detected (Pascoe et al., 1992b).
These findings suggest that exon 5 of CYP11B2 is essential
in conferring aldosterone synthase activity to CYP11B1. A
chimera consisting of the promotor and exon 1 to exon 4
of CYP11B2 and exon 5 to exon 9 of CYP11B1 was found
in a patient suffering from congenital adrenal hyperplasia,
due to insufficient expression of this gene resulting from
the replacement of the strong ACTH-inducible promoter by
the regulatory elements of CYP11B2 (Hampf et al., 2001).
5. Regulation on the molecular level
5.1. Activity modulation by the primary structure
Valuable clues for the identification of important residues
in human CYP11B enzymes came from the analysis of
 M. Lisurek, R. Bernhardt / Molecular and Cellular Endocrinology 215 (2004) 149–159 153

Table 1
List of published missense mutations in the human CYP11B1 gene
Mutation Patient Exon Putative 3D location Effect in vitro Reference

Pro42Ser Female girl 1 A-helix 15% reduced 11␤-hydroxylase activity Joehrer et al. (1997)
Val129Met Caucasian, male 2 C-helix Complete loss of 11␤-hydroxylase activity Geley et al. (1996)
Asn133His Female girl 3 C-helix 17% reduced 11␤-hydroxylase activity Joehrer et al. (1997)
Thr318Met Yemenite, female 5 I-helix, active site Complete loss of 11␤-hydroxylase activity Curnow et al. (1993)
Thr319Met Female girl 6 I-helix, active site 37% reduced 11␤-hydroxylase activity Joehrer et al. (1997)
Ala331Val Caucasian, female 6 I-helix Complete loss of 11␤-hydroxylase activity Geley et al. (1996)
Glu371Gly Caucasian, female 6 K-helix Complete loss of 11␤-hydroxylase activity Geley et al. (1996)
Arg374Gln Lebanese Arab, female 6 K-helix Complete loss of 11␤-hydroxylase activity Curnow et al. (1993)
Arg384Gln Adult female 7 Heme propionate neutralisation Complete loss of 11␤-hydroxylase activity Curnow et al. (1993)
Val441Glu Adult male 8 Meander Complete loss of 11␤-hydroxylase activity Curnow et al. (1993)
Arg448Cys Iranian, male 8 Heme propionate neutralisation Complete loss of 11␤-hydroxylase activity Geley et al. (1996)
Arg448His Moroccan Jew 8 Heme propionate neutralisation Complete loss of 11␤-hydroxylase activity White et al. (1991)
The mutations were found in patients suffering from CAH and were subsequently demonstrated to lead to abolished 11␤-hydroxylase activity in cell
culture experiments.

patients suffering from genetic aberrations in the CYP11B1
and CYP11B2 genes.
11␤-Hydroxylase deficiency, besides 21-hydroxylase de-
ficiency, is the second most common cause of CAH, an in-
herited disease with the inability to synthesise cortisol from
11-deoxycorticosterone. Characteristically this disease leads
to androgen excess and hypertension. Besides the mutations
mentioned above, many other missense mutations which
have been found in patients suffering from this disease, lead
to functionally disturbed enzymes after expression in cell
culture (see Table 1). Curnow et al. (1993) demonstrated that
mutations T318M, R374Q, R384Q and V441G in exons 5,
6, 7 and 8 lead to complete dysfunctional CYP11B1 activity.
Mutations V129M, A331V, E371G and R448C were shown
by Geley et al. (1996) to be defective for 11␤-hydroxylase
activity. Mutations P42S, N133H and T319M lead only to
partially disturbed 11␤-hydroxylase activity (Joehrer et al.,
1997). As can be seen in Table 1, mutations leading to
11␤-hydroxylase deficiency are distributed over the en-
tire coding region, but with a slightly enhanced frequency
in exons 6 and 8. This might reflect the presence of
functionally important amino acid residues in these re-
gions or, alternatively, mutations in this region are more
likely to have deleterious effects on enzymatic activity.
Amino acid residue P42 in the A-helix is speculated to
be essential for proper membrane orientation (Joehrer
et al., 1997). Amino acids V129 and A331 are highly con-
served among members of the CYP11B family, but not
between CYP11B1 and other cytochrome P450 enzymes.
This is an indirect indication of the functional importance
of these amino acids, but the precise reason for their dis-
ruptive effects remains to be elucidated. Amino acid N133
is also conserved among members of the CYP11B family
and seems to lie within the region that may form part of
the substrate access channel (Joehrer et al., 1997). The con-
served homologous residue to T318 in human CYP11B1 is
T252 in CYP101 and this residue has been shown to be in-
volved in proton transfer which is necessary for enzymatic
activity (Raag et al., 1991; Ravichandran et al., 1993). The
effect of mutations E371 and R374 in the K-helix may be
well rationalised by the fact that these residues, highly con-
served among most cytochrome P450 families form part of
the ERR-triad and are thought to play a fundamental role
in enzyme stabilisation (Hasemann et al., 1995).
Isolated deficiencies of aldosterone biosynthesis caused
by CYP11B2 gene defects (see Table 2) are called aldos-
terone synthase deficiencies type I and type II, formerly
called corticosterone methyl oxidase (CMO) deficiencies,
which lead to hypoaldosteronism. Since the nomenclature
and classification of these different diseases is being dis-
cussed controversially (Portrat-Doyen et al., 1998), they
will be herein referred to only as aldosterone synthase

Table 2
List of published missense mutations in the human CYP11B2 gene
Mutation Patient Exon Putative 3D location Effect in vitro Reference

Thr185Ile Boy 3 E-helix Reduced 18-hydroxylase activity, no detectable Peter et al. (1998), Hampf
18-oxidase activity et al., unpublished results
Arg384Pro Caucasian, male 7 ␤1-4 Complete loss of aldosterone synthase activity Geley et al. (1995)
Leu461Pro Turk 8 L-helix Complete loss of aldosterone synthase activity Nomoto et al. (1997)
Val386Ala/Arg181Trp Iranian Jew 7/3 ␤1-4/between Increased 11␤- and 18-hydroxylase activity, no Pascoe et al. (1992a)
D- and E-helix detectable 18-oxidase activity
Val386Ala/Glu198Asp French twin boy 7/3 ␤1-4/E-helix Increased 11␤- and 18-hydroxylase activity, no Portrat-Doyen et al. (1998)
detectable 18-oxidase activity
The mutations were found in patients suffering from aldosterone synthase deficiency and were subsequently demonstrated to lead to abolished aldosterone
synthase activity in cell culture experiments.
154 M. Lisurek, R. Bernhardt / Molecular and Cellular Endocrinology 215 (2004) 149–159
deficiencies. These disorders lead to decreased sodium re-
sorption and potassium secretion to the urine. The plasma
renin activity is elevated and the plasma levels of aldos-
terone precursors are elevated. All patients suffered in
infancy from failure to thrive. The mutation Thr185Ile has
been found in a boy suffering from aldosterone synthase
deficiency (Peter et al., 1998), and subsequent investiga-
tions in our laboratory showed that this mutation leads to
a severe reduction of 18-hydroxylation and to a complete
loss of aldosterone synthase activity (Hampf et al., un-
published results). Although CYP11B1 and CYP11B2 are
invariant at this position, the mutated CYP11B2 enzyme
displayed much stronger 11␤-hydroxylase activity than
wild type CYP11B2 towards both 11-deoxycorticosterone
and 11-deoxycortisol. The mutation Arg384Pro leads to al-
dosterone deficiency (Geley et al., 1995). Double mutations
Glu198Asp and Val386Ala lead to enzymes with decreased
11␤- and 18-hydroxylase activity and impaired 18-oxidase
activity (Portrat-Doyen et al., 1998), however, none of
these mutations alone abolished aldosterone synthase ac-
tivity completely. Mutations Arg181Trp and Val386Ala
were found in Iranian Jews suffering from aldosterone
deficiency. Subsequent analysis of the CYP11B2 genes
in COS-1 cells showed that Arg181Trp does not affect
11␤-hydroxylation activity, but reduces 18-hydroxylation
and 18-oxidation activity. Additional presence of Val386Ala
abolishes 18-oxidation activity completely (Pascoe et al.,
1992a). Taken together, it can be quoted that aldosterone
synthase deficiencies due to missense mutations are less
frequent than 11␤-hydroxylation deficiencies and that the
role of these highly conserved residues remains to be elu-
cidated. A variety of mutations found in patients that suffer
from 11␤-hydroxylase and aldosterone synthase deficien-
cies, like point mutations, deletions and incorrect splicing in
the CYP11B1 and CYP11B2 genes leading to completely
inactive enzymes are summarised by Peter et al. (1999).
In an effort to identify the minimum number of mutations
relevant for the different specific hydroxylation reactions be-
tween the CYP11B-family members, residue-swapping mu-
tants between these isoenzymes have been investigated us-
ing site-directed mutagenesis.
Residues differing between human CYP11B1 and
CYP11B2 localised in the N-terminal part of the enzymes
have been investigated by Bechtel et al. (2002) by transient
expression of CYP11B2 mutants in COS-1 cells. The study
revealed a three-fold increased 11␤-hydroxylation after
substitution of Ile112 by proline and an even a four-fold in-
creased activity is exhibited by mutant Asp147Glu. A com-
bination of both mutations increased the 11␤-hydroxylation
activity towards 11-deoxycorticosterone six-fold. The ef-
fect on the second and third catalytic step of CYP11B2
was less pronounced, except for Ile112Pro which showed
an about two-fold increased 18-hydroxylation activity
(Fig. 5). Replacements Ile112Ser and Lys152Asn did not
alter CYP11B2 substrate conversion. These investigations
demonstrated that the three enzymatic reaction steps of
Fig. 5. Presentation of relative aldosterone synthase activities of wild
type CYP11B2 and effect of the mutants Ile112Pro, Asp147Glu and
Ile112Pro/Asp147Glu on 11␤-hydroxylation, 18-hydroxylation and 18-ox-
idation towards 11-deoxycorticosterone (DOC) expressed in COS-1 cells.
aldosterone synthesis may be modified independently, af-
fecting only one reaction step, or differentially by altering
the three hydroxylation steps to distinct degrees.
The effect of the mutation Lys251Arg in CYP11B2
was investigated by Fardella et al. (1995). It caused four
times as much 18-hydroxycorticosterone formation and
50–80% increased aldosterone formation. If such a muta-
tion would occur in patients, the predicted phenotype would
be low-renin hypertension, due to the overproduction of
aldosterone caused by this CYP11B2 mutant.
Very interesting activity swapping mutants have been
obtained by mutating amino acids located in the putative
I-helix. It was found that single mutations at positions 296,
301, 302, 320, and 335 in CYP11B2 exhibited only slightly
increased 11␤-hydroxylase activities in CYP11B2 when
exchanged to the corresponding ones found in CYP11B1
(Böttner et al., 1996). However, a Leu301Pro/Ala320Val
double substitution increased 11␤-hydroxylase activity
to 60% as compared to that of wild type CYP11B1,
and the additional substitution Glu302Asp further en-
hanced this activity to 85%. Concomitantly, the aldos-
terone synthase activities of the mutant CYP11B2 pro-
teins were suppressed to varying degrees, with double
and triple replacement mutants Leu301Pro/Ala320Val and
Leu301Pro/Glu302Asp/Ala320Val retaining only about
10% of CYP11B2 wild type activity (Fig. 6). Thus, a
mineralocorticoid-synthesising P450 has been engineered to
a glucocorticoid-synthesising enzyme by only two to three
point mutations (Böttner et al., 1996). In reciprocal residue
swapping experiments, CYP11B1 mutant Val320Ala, either
alone (Böttner and Bernhardt, 1996) (Fig. 6) or in com-
bination with mutation Ser288Gly (Curnow et al., 1997;
Mulatero et al., 1998) or Asn335Asp (Böttner et al., 1998),
conferred aldosterone synthase activity to CYP11B1, para-
doxically without significantly affecting 11␤-hydroxylase
activity. Thus, a glucocorticoid-synthesising enzyme has
 M. Lisurek, R. Bernhardt / Molecular and Cellular Endocrinology 215 (2004) 149–159
Fig. 6. Presentation of aldosterone and cortisol formation of wild
type human CYP11B2 and CYP11B1. CYP11B2, the mineralocorti-
coid synthesising protein has been changed to a glucocorticoid syn-
thesising one by two or three point mutations (Leu301Pro/Ala320Val
and Leu301Pro/Glu302Asp/Ala320Val). CYP11B1 became an aldosterone
synthase by a single point mutation (Val320Ala).
been engineered to a mineralocorticoid-synthesising en-
zyme by a single point mutation.
Diverging residues in the C-terminal regions of CYP11B1
and CYP11B2 at positions 471, 472, 492, 493 and 494,
have been investigated, but were shown to be insignificant
for the stereo- and regiospecificity of steroid hydroxyla-
tion (Böttner et al., 1998). These investigations, in combi-
nation with naturally occurring chimeras of CYP11B1 and
CYP11B2, suggested that there are residues spread through-
out the protein which are important for proper stereoselec-
tive hydroxylation activity. However, the residue swapping
experiments suggest that side chains located in the puta-
tive I-helix of the CYP11B enzymes contribute most signif-
icantly to substrate recognition and conversion. The I-helix
is translated from exon 5 and exon 6 which further un-
derlines the findings of Pascoe et al. (1992b) that exon 5
155
is essential in conferring aldosterone synthase activity to
CYP11B1.
5.2. Activity modulation of CYP11B1 and CYP11B2 by
regulation of the electron transfer rate
As mentioned above, mitochondrial cytochrome P450-
dependent steroid hydroxylases require a NADPH-dependent
redox system consisting of adrenodoxin reductase and
adrenodoxin (Adx) (Bernhardt, 1996). It has been shown
that positively charged residues on the proximal surface
of the cytochrome P450 on the one hand (Wada and
Waterman, 1992; Lepesheva et al., 2000; Azeva et al.,
2001) and both negatively charged amino acids (Coghlan
and Vickery, 1991, 1992) as well as hydrophobic residues
on the surface of Adx on the other hand (Beckert et al.,
1994; Beckert and Bernhardt, 1997; Bernhardt, 1998) are
important for the correct function of the ferredoxin as an
efficient electron carrier.
In COS-1 cells the source of reducing equivalents is
limited. The activity of the bovine and human CYP11B
isoenzymes is stimulated by an enhanced electron trans-
fer due to an increased expression of cotransfected Adx
(Zuber et al., 1988; Mathew et al., 1990). In addition,
in case of bovine CYP11B1 the ratio of corticosterone
to 18-hydroxycorticosterone and to aldosterone decreases
with a high level of cotransfected Adx. This is also the
case for human CYP11B2. Cotransfected Adx stimulated
18-hydroxylation and 18-oxidation activity besides in-
creasing 11␤-hydroxylation activity. This indicates that the
availability of a sufficient amount of electrons supports
the formation of 18-hydroxycorticosterone and aldosterone
(Cao and Bernhardt, 1999b).
The effects caused by truncated forms of Adx (e.g. Adx
4-108 and Adx 4-114) have been investigated with respect
to their reduction rate of CYP11B1. The truncated mutants
Adx 4-108 and Adx 4-114 led to an increase in corti-
costerone formation from 11-deoxycorticosterone using a
liposomal reconstitution assay with bovine CYP11B1 (Cao
and Bernhardt, 1999a). The apparent Vmax values of al-
dosterone formation by bovine CYP11B1 were determined
to be three-fold higher for Adx 4-108 and two-fold higher
for Adx 4-114 as compared to wild type Adx. Subsequent
investigations in COS-1 cells confirmed the stimulating
effect of cotransfected Adx 1-108 on 11␤-hydroxylation
activity of bovine CYP11B1, human CYP11B1 and human
CYP11B2 as well as an enhancement of 18-hydroxylation
and 18-oxidation activity of bovine CYP11B1 and human
CYP11B2 as compared to cotransfection with wild type Adx
(Cao and Bernhardt, 1999a). Both 18-hydroxycorticosterone
and aldosterone are correlated with hypertension. Several
C-terminally truncated species of Adx, e.g. species with 114,
121, 124, 125 and 127 amino acid residues, respectively,
have been isolated from bovine adrenal cortex (Tanaka et al.,
1973; Hiwatashi et al., 1986; Sakihama et al., 1988). Un-
der circumstances where Adx is progressively C-terminally
156 M. Lisurek, R. Bernhardt / Molecular and Cellular Endocrinology 215 (2004) 149–159
truncated in vivo the accompanying increased electron
transfer ability of the truncated Adx could lead to regulatory
relevance in a way that the respective adrenal glands would
secrete more aldosterone (Uhlmann et al., 1994). However,
it is not known whether the truncated species of Adx are
artefacts or whether physiological conditions leading to a
changed electron flow in adrenal tissue do occur.
Taken togeher, the results obtained with different Adx
mutants, which display different electron transfer properties
support the proposal that an increase in the electron transfer
rate leads to an enhancement of the ‘intermediate flow’ to-
wards aldosterone. This observation could be of physiologi-
cal importance since in bovine adrenal cortex the molar ratio
of AdR/Adx/CYP11B1 is approximately 1:3:4 (Hanukoglu
and Hanukoglu, 1986). The corresponding ratio in human
tissue has not been determined. An increase of the concen-
tration of wild type Adx, e.g. in the case of ACTH-dependent
induction, could exert a significant effect on aldosterone pro-
duction under these conditions.
5.3. Activity modulation by protein–protein interaction
with CYP11A1
The aspect of activity modulation of CYP11B enzymes
by interaction with CYP11A1, the cholesterol side chain
cleavage enzyme, which is the only other mitochondrial
P450 in the adrenal cortex, has been studied intensively.
Competition between rat CYP11A1 and CYP11B1 for re-
ducing equivalents is observed in vitro (Yamazaki et al.,
1993). Additionally, transfection with the human or bovine
enzymes in COS-1 cells containing a limited amount of
reducing equivalents resulted in a decrease of cortisol and
aldosterone synthesis, respectively (Cao and Bernhardt,
1999b). In contrast to this, the bovine enzymes displayed
a specific interaction indicating allosteric effects of bovine
CYP11A1 on bovine CYP11B1 in reconstituted liposo-
mal membranes (Ikushiro et al., 1992; Kominami et al.,
1994) and in COS-1 cells capable of high electron transfer
rates due to cotransfection of Adx (Cao and Bernhardt,
1999b). In the liposomal membranes and in COS-1 cells
bovine CYP11A1 stimulated 11␤-hydroxylation activity of
bovine CYP11B1, and decreased 18-hydroxycorticosterone
and aldosterone formation at the same time. Therefore, it
is tempting to speculate that CYP11A1 induces confor-
mational changes in CYP11B1 which differentially affect
binding of the substrate and the intermediates, so that
corticosterone can leave the active site in favour of being
further 18-hydroxylated and 18-oxidised. However, human
CYP11A1 does not show such a specific interaction with
human CYP11B2. There is only competition for reducing
equivalents at low concentrations of electrons supplied and
an increase in all enzymatic activities at high electron sup-
ply, but no effect on the product pattern (Cao and Bernhardt,
1999b).
Modulation of CYP11B2 activity has also been shown to
depend on other circumstances. Aldosterone formation may
be differentially regulated on the level of membrane compo-
sition: in a phospholipid vesicle system, a strong dependence
of aldosterone synthesis on the composition of the lipids and
the presence of calmodulin was observed (Ohnishi et al.,
1984, 1986; Wada et al., 1984; Seybert, 1990). Another hy-
pothesis suggests that CYP11B2 activity may be controlled
post-translationally by protein phosphorylation producing
short-term activity control (Bureik et al., 2002b). Further
studies are, however, necessary to support this finding.
6. Outlook
Overexpression of CYP11B1 and CYP11B2 in bacteria
has not been successful so far. Only expression of the rat
homologues was achieved at low yield in Escherichia coli
(Nonaka et al., 1998). Besides in mammalian cells the func-
tional expression of human CYP11B1 and CYP11B2 using
the fission yeast Schizosaccharomyces pombe (Bureik et al.,
2002c) was demonstrated. It was found that the transformed
yeast show strong steroid hydroxylase activity in vivo in
comparison to transfected mammalian cells (see above). So
far, most studies performed with these enzymes used recom-
binant cell cultures, and therefore detailed studies on the
biochemical reaction mechanism and properties (e.g. sub-
strate binding, protein–protein interactions, electron trans-
fer, substrate induced structural changes and EPR spectra)
are not available. It can be expected that development in
this field will allow the production and isolation of human
CYP11Bs and their mutants and improve our knowledge on
the structure–function relationship of these highly homolo-
gous but nevertheless diverse enzymes.
As shown above, different mechanisms may lead to over-
production of aldosterone. A pathologically high level of
plasma aldosterone, on the other hand, may not only lead to
hypertension, but could also cause progression of congestive
heart failure and myocardial fibrosis, as demonstrated re-
cently. Treatment with antagonists of the mineralocorticoid
receptor leads to decreased morbidity and mortality of the
patients (Pitt, 2002), but is accompanied by severe side ef-
fects. Thus, inhibition of aldosterone production by inhibit-
ing CYP11B2 might be a more sophisticated approach to
tackle this problem. Initial studies using recombinant V79
and S. pombe cells showed that it is possible to find selec-
tive CYP11B inhibitors (Denner et al., 1995; Denner and
Bernhardt, 1998; Ehmer et al., 2002), which could interfere
with the fatal processes of mineralocorticoid receptor an-
tagonists. An overview about the recent development of in-
hibitors of both, CYP11B1 and CYP11B2, is given in Bureik
et al. (2002a).
Acknowledgements
We thank Matthias Bureik and Andy Zöllner for critical
reading of the manuscript.
 M. Lisurek, R. Bernhardt / Molecular and Cellular Endocrinology 215 (2004) 149–159
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