(Received for publication, October 31, 1995; and in revised form, January 19, 1996)
From the
Site-directed mutagenesis of a domain (amino acids
299-338) aligning to the I-helix region of P450,
P450
and P450
was used to investigate the
different regioselectivities displayed in the hydroxylation reactions
performed by human aldosterone synthase (P450
) and
11
-hydroxylase (P450
). The two enzymes are 93%
identical and are essential for the synthesis of mineralocorticoids and
glucocorticoids in the human adrenal gland. Single replacement of
P450
residues for P450
-specific
residues at positions 296, 301, 302, 320, and 335 only gave rise to
slightly increased 11
-hydroxylase activities. However, a
L301P/A320V double substitution increased 11
-hydroxylase activity
to 60% as compared with that of P450
. Additionally
substituting Ala-320 for Val-320 of P450
further
enhanced this activity to 85%. The aldosterone synthase activities of
the mutant P450
proteins were suppressed to a varying
degree, with triple replacement mutant L301P/E302D/A320V retaining only
10% and double replacement mutant L301P/A320V retaining only 13% of the
P450
wild type activity. These results demonstrate a
switch in regio- and stereoselectivities of the engineered
P450
enzyme due to manipulation of residues at three
critical positions, and we attribute the determination of these
features in P450
to the structure of a region analogous
to the I-helix in P450
.
In the adrenal gland essential steroid hormones such as
glucocorticoids, mineralocorticoids, and androgens are produced.
Cortisol, the major glucocorticoid in humans, is synthesized in the
zona fasciculata/reticularis under control of pituitary derived
adrenocorticotropic hormone, whereas the most potent mineralocorticoid,
aldosterone, is secreted from zona glomerulosa cells primarily in
response to angiotensin II and
potassium(1, 2, 3) . This differential
secretion is achieved by a diverging expression pattern of a series of
monooxygenases, also called P450 ()enzymes, which catalyze a
multistep process providing the organism with the effective hormones.
In humans the final steps in cortisol and aldosterone production,
precisely 11
-hydroxylation in the zona fasciculata/reticularis and
11
-hydroxylation, 18-hydroxylation, and 18-oxidation in the zona
glomerulosa, are performed by two distinct enzymes, namely
11
-hydroxylase (P450
) and aldosterone synthase
(P450
). The genes encoding these enzymes, CYP11B1 (P450
) and CYP11B2 (P450
), have been isolated from a genomic library (4) and were shown by sequence comparison to be members of the
superfamily of cytochrome P450 genes(5) . Further structural
characterization revealed nine exons spanning the genes, which are
tandemly arranged on chromosome 8q22(6, 7) . The
molecular masses of the respective proteins have been determined to be
50 (P450
) and 48.5 (P450
)
kDa(8) . Both enzymes, after being synthesized, are
translocated into the mitochondrial matrix where they are bound to the
inner membrane by as yet not precisely defined segments of the
proteins. There, accompanied by an NADPH-dependent redox system
consisting of a flavoprotein, adrenodoxin reductase, and an iron-sulfur
protein, adrenodoxin, donating reducing equivalents, they participate
in steroid hydroxylation. P450
enzymes of other
species have been extensively studied, and it turned out that in
bovine(9) , porcine (10) , and frog (11) adrenal cortex, synthesis of gluco- and mineralocorticoids
is catalyzed by a single enzyme. Conversely, synthesis of human,
rat(12) , and mouse (13) gluco- and mineralocorticoids
have been separated in evolution and are carried out by distinct
enzymes, yet the reason for these interspecies differences is
enigmatic. We intended to gain insight into the principles underlying
the different regioselectivities involved in 11
-hydroxylation and
18-hydroxylation/oxidation in the human enzymes. Since both proteins
are 93% identical yet carry out separate reactions to yield different
steroid hormones, it remained elusive on which structure-function
relationships these diversities could be based. Recently, the cause of
glucocorticoid-remediable aldosteronism, an autosomal dominant disorder
in humans, was reported to arise from unequal crossing-over events
between the CYP11B1 and CYP11B2 genes(14) .
The resulting chimeric genes comprise a 5` CYP11B1 portion and
a 3` CYP11B2 portion under control of the CYP11B1 regulatory region. Pascoe et al.(15) , through
the analyses of hybrid proteins, determined the C-terminal 247 amino
acids of P450
as crucial for aldosterone synthesis.
Keeping this in mind, we carried out a computer-based sequence and
structure alignment with three of the four by now crystallized P450
proteins, namely P450
from Pseudomonas
putida(16) , P450
from Bacillus
megaterium(17) , and P450
from another Pseudomonas species(18) . We performed site-directed
mutagenesis on a region supposedly analogous to the P450
I-helix and subsequent analyses of the mutants by transient
transfection experiments using COS-1 cells. This led to the
identification of mutant P450
proteins having
dramatically increased 11
-hydroxylase activity, which in the
P450
wild type protein is considerably lower than in the
P450
wild type protein(19) . Concomitantly,
aldosterone synthase activity in these mutants was lost to a
substantial degree, indicating that regioselectivities have
successfully been switched from one position to the other.
Oligonucleotides were synthesized on an Applied Biosystems model
380A DNA synthesizer at BioTez (Berlin). Restriction enzymes, Klenow
fragment of DNA polymerase I, T4 polynucleotide kinase, bovine alkaline
phosphatase, T4 DNA ligase, and DH5 cells were purchased from New
England Biolabs Inc. or Boehringer Mannheim. Taq polymerase
was obtained from Perkin-Elmer. pALTER-1 plasmid, helper phage R408,
JM109, and ES1301 mutS cells were obtained as part of the
Altered Sites in vitro mutagenesis system (Promega Co.,
Madison, WI). pRc/CMV was from Invitrogen, and pBS SK(+) was from
Stratagene. The
Taq
cycle sequencing kit
was purchased from U.S. Biochemical Corp., and
[
S]dATP was from Amersham Corp.
[
H]Deoxycorticosterone and
[
H]deoxycortisol were obtained from DuPont NEN.
Deoxycorticosterone, corticosterone, deoxycortisol, cortisol,
chloroquine, cell culture-tested HEPES, and dimethyl sulfoxide were
purchased from Sigma. DEAE-dextran was purchased from Pharmacia Biotech
Inc. Radioimmunoassays were performed with Active
coated
radioimmunoassay kits from Diagnostic System Laboratories Inc. ECL
Western blotting reagents were obtained from Amersham, polyclonal
hemagglutinin (HA) 11 antibody was from the BAbCO Berkeley Antibody
Company, and the polyclonal anti-bovine adrenodoxin (Adx) antibody was
raised in rabbit by Eurogentec
. Nitrocellulose membrane
was used from Schleicher & Schuell.
Alternatively,
steroids were measured using an Active aldosterone or
Active
cortisol radioimmunoassay.
Figure 1:
Comparison
of the human P450 and P450
sequences
with those of bacterial P450
, P450
, and
P450
. Residues predicted to be contained in the putative
I-helix are in white on black, and those positions (296, 301,
302, 320, 335, and 339) varying between the human P450
and P450
sequences in this region are printed
in boldface type. The P450
and P450
were aligned to P450
as described under
``Experimental Procedures,'' and the P450
and
P450
are aligned with P450
as described by
Ravichandran et al. (17) and Hasemann et
al.(18) . In addition we included the sequences of the
human P450
and P450
in the
alignment.
To
investigate the role of the putative I-helix in the regioselectivity of
P450- and P450
-specific hydroxylation
reactions, we performed site-directed mutagenesis on the P450
cDNA. Derived from the alignment studies, the region
corresponding to the I-helix in P450
extends from amino
acid 299 to 338 (Fig. 2). This part of the total sequence
includes four diverging amino acids at positions 301, 302, 320, and
335, but we also analyzed the role of Lys-296 in our investigations,
since also a flanking amino acid could exert an influence on the
positioning of the putative I-helix and thereby the regiospecificity of
hydroxylation. Mutants were generated using the mutagenic
oligonucleotides listed in Table 1. Thus,
P450
-specific amino acid residues were substituted for
P450
-specific ones, and single, double, and triple
replacement mutants of P450
were created.
Figure 2:
Schematic representation of the tagged
P450 and P450
proteins. The HA tag is
represented by a solid black box, and the putative I-helix is
represented by a striped box. The numbering at the bottom represents the amino acid positions of the coding
regions of the proteins. The precise peptide sequences of the predicted
I-helix positions 299-338 are shown at the top; the
proteins encoding them are labeled on the right side. Amino
acids differing between the two proteins are printed in boldface
type. P450
residues at positions 296, 301, 302,
320, and 335 were mutated to P450
residues, and the
HA epitope was fused to the C termini of the proteins as described
under ``Experimental
Procedures.''
Figure 3:
HPLC analysis of 11-hydroxylase
activities of P450
mutants. Transfected COS-1 cells were
incubated with [
H]11-deoxycortisol for 48 h.
Steroids were extracted out of the medium analyzed by normal phase
HPLC. Under the given experimental conditions 11
-hydroxylase
activity of P450
was not detectable. Activities of the
mutants L301P, L301P/D302E, L301P/A302V, and L301P/D302E/A320V were
calculated by integration of the peaks and mounted up to 4, 10, 60, and
80% as compared with the P450
activity and thus
corresponded well to the results shown in Fig. 4A. Boxes on the top denote the positions of
11-deoxycortisol (S) and cortisol (C).
Figure 4:
A, 11-hydroxylase activities of
P450
mutants expressed in COS-1 cells. Cells transfected
with the indicated mutant or wild type cDNAs were incubated with 5
µM 11-deoxycortisol for 24 h. The amount of cortisol
arisen from the reaction was analyzed by radioimmunoassay. Plotted
values with standard errors represent the means of four
independent transfections for each of the proteins and are normalized
against the activity of the P450
wild type. Mutants
are designated at the bottom of the panel. B, the same experimental design as described for panel A was used, except that cells were incubated with 5 µM 11-deoxycorticosterone and finally assayed for aldosterone arisen
from the conversion.
Because of the dual ability of the
P450 wild type enzyme to produce both cortisol and
aldosterone in vitro we investigated aldosterone synthase
activity in this set of P450
mutants. The primary
question was whether one of the two activities (cortisol synthesis)
could be increased without affecting the other activity (aldosterone
synthesis) or whether there is a reciprocal behavior to be found,
meaning no increase in one activity without loss in the other.
Examination of the aldosterone-synthesizing abilities of the mutants
showed that triple mutant L301P/E302D/A320V only retained about 10% and
double mutant L301P/A320V about 13% of the P450
wild
type activity (Fig. 4B). Mutants L301P/E302D and A320V
were only slightly compromised in their activities, but the combination
of both resulting in the triple mutant synergize drastically to a
severe loss in 18-hydroxylation or 18-oxidation activity.
Paradoxically, double mutant L301P/E302D was less severely affected in
its aldosterone-synthesizing capacity (87% retained as compared with
P450
wild type) than the respective single mutants.
Introducing P450
residues at positions 296 and 335
decreased activity to about 40% in the single mutants. The combined
double mutant is reduced to about 15% in its aldosterone-synthesizing
capacity as compared with P450
wild type, and
additionally substituting Asp at position 335 for Asn totally destroys
aldosterone synthase activity. These data show that every alteration
made in this region did negatively affect the aldosterone synthase
activity of the recombinant enzyme although to a varying degree.
Figure 5:
Immunological detection of heterologously
expressed P450 mutants in COS-1 cells. Total proteins
were separated on an SDS gel, followed by electroblotting to a
nitrocellulose filter. P450
wild type, P450
wild type, and P450
mutant proteins were detected
with a rabbit polyclonal anti-HA-directed antibody (upper
panels), and bovine adrenodoxin was detected with a rabbit
anti-BAdx antibody (lower panel). A peroxidase-conjugated
secondary antibody and ECL chemiluminescence served for visualization
of bands. A, detection of single replacement mutants in
comparison with P450
wild type and P450
wild type proteins. B, detection of multiple replacement
mutants in comparison with P450
wild type and
P450
wild type proteins.
In pursuing structure to function relationships in mammalian
P450 proteins, alignment to the bacterial P450 and
recently also to P450
and P450
has proved
to be a useful approach. However, a relatively low amino acid sequence
homology often hampers accurate alignment of specific
residues(33) . We therefore benefited from the HOMOL data base
developed by D. Nelson, which combines amino acid identities and
secondary structure predictions. By including in our alignment also the
P450
and P450
sequences, which (due to the
revealed secondary structures) could be structurally aligned to
P450
(17, 18) , we intended to improve
the prediction of structural entities in P450
and
P450
. Since for P450
it is well
established that the I-helix critically participates in substrate
binding and because the P450
and P450
proteins show some diversities in a region corresponding to the
P450
I-helix, we hypothesized that these differences
could be the basis for the different regioselectivities of the two
steroid hydroxylases. The I-helix in P450
, like in
P450
and P450
, runs like a tube through
the interior of the molecule (Fig. 6) and in the case of
P450
, together with the heme binding region, makes up
part of the substrate binding pocket(16, 34) . One
critical feature of this conserved helix in P450 proteins is Thr-252 in
P450
, necessary for the activation of molecular oxygen (28, 29, 30) . In P450
the
substrate camphor is found to be in tight association with the -VGGL-
stretch, where the two Gly residues induce a bend in the helix and thus
serve as a site where the substrate can fit into position correctly.
There is only one Gly residue of the -VGGL- motif conserved in
P450
and P450
(Fig. 1) and it
is questionable whether this is sufficient for bending the helix
likewise.
Figure 6:
Model of the P450 and
P450
structure. Three-dimensional structure of the
entire P450
and P450
structures with
the incorporated heme is shown. The view is focused onto the putative
I-helix region, and amino acid residues varying between P450
and P450
are marked in black.
A possible relevance of the putative I-helix region for
the regioselectivity is also supported by the observations of Pascoe
and colleagues(15) . By studying artificially engineered hybrid
proteins with variable N-terminal P450 and C-terminal
P450
portions reflecting an in vitro model of
glucocorticoid-remediable aldosteronism, a genetic disorder, His-256,
was defined as a critical breakpoint, and the sequence C-terminal of it
was identified as essential for aldosterone synthesis. In performing
site-directed mutagenesis on the P450
protein and
assessing 11
-hydroxylase activities in transfected COS-1 cells, we
found that single amino acid replacements by
P450
-specific residues at positions 296 and 335 only
slightly increased 11
-hydroxylase actvity and that there was up to
a 2-fold increase detected when position 301, 302, or 320 harbored
P450
residues. However, double substitution
L301P/E302D already conferred 60%, and triple replacement mutant
L301P/E302D/A320V mounted up to an activity being about 85%, that of
the P450
wild type protein, given that under these
experimental conditions the P450
wild type enzyme
exhibited only 5% of the activity of the P450
wild
type (Fig. 4A). These data indicate a synergistic
rather than a mere additive effect contributed by these three residues.
A nonconservative change from Leu to Pro at position 301 alone had no
substantial impact, although it was expected to exert other than size
effects, since Pro in many cases distorts if not destroys helix
continuity. In contrast, the overall effect on cortisol production
could be drastically enhanced by two additional conservative
substitutions at positions 302 and 320, suggesting that the size of the
side chains at these positions is critical for 11
-hydroxylase
activity.
In assaying the aldosterone-synthesizing activities of the
mutants, we found mildly to strongly decreased activities.
Interestingly, already single substitutions in some mutants markedly
reduced aldosterone synthesis. Contrasting 11-hydroxylase
activity, P450
residues Lys-296 and Asp-335 obviously
are important for maximum aldosterone synthase activity since single
substitution at these positions to Asn lowered aldosterone synthesis to
about 40%. Moreover, double replacement mutant L296N/D335N showed an
aldosterone synthase activity decreased to 15%, which was completely
abolished by adding the A320V mutation. Among the mutants still
producing aldosterone, double replacement mutant L301P/A320V and triple
replacement mutant L301P/E302D/A320V gave rise to the most reduced
aldosterone levels (13 and 10% of the P450
wild type),
which reciprocally parallels their increase in 11
-hydroxylase
activity. In conclusion, there is no strict correlation between
11
-hydroxylase increase and decrease in aldosterone synthesis to
be seen in this set of mutants. Whereas Pro-301, Asp-302, and Val-320
clearly are major contributors to 11
-hydroxylation, aldosterone
synthesis, apart from the residues at these positions in the
P450
sequence also seems to be influenced by Lys-296 and
Asp-335. Aldosterone synthesis thus is dependent on a highly evolved
structure in the P450
protein and is susceptible to
minor changes in this region.
Until now, no CYP11B2 defects have been linked to this region in patients suffering from hypoaldosteronism, but it is conceivable that their occurrence would deteriorate mineralocorticoid synthesis. Whether the positions targeted by site-directed mutagenesis directly contact the substrate or decisively alter the position or structure of the I-helix remains an open topic.
The I-helix also was postulated in modelling studies of
P450 by Graham-Lorence and co-workers (35) and
P450
by Laughton and co-workers (36) to form
part of the substrate binding pocket for the substrates androstenedion
and pregnenolone or progesterone, respectively. Since these are also
steroid-modifying enzymes, one is tempted to draw a general
significance for this structure in steroid hydroxylation.
In
contrast, for P450 proteins that belong to the CYP2 family and
participate in liver microsomal steroid hydroxylation, it has been
shown that also the N-terminal part of these proteins contributes to
the substrate specificities and regioselectivities of hydroxylations
(reviewed in (37) and (38) ). Lindberg and Negeshi (39) have shown that only one substitution, F209L, was
sufficient to confer steroid 15-hydroxylase activity from P450 2A4
to P450 2A5, a coumarin hydroxylase. Recently, Halpert and He (40) were able to shift P450 2B1 androgen hydroxylation between
the 15
- and 16
-positions by manipulating two positions,
namely Ile-114 and Gly-478, of the enzyme. Moreover, progesterone
21-hydroxylation of P450 2C4 and P450 2C5 seems to be dependent on a
hypervariable N-terminal region (residues 95-123) of the proteins
and could be conferred on P450 2C1 (41) by mutating the same
region of the otherwise nonactive protein in 21-hydroxylation. The same
activity could be generated by the creation of chimeric proteins
composed of P450 2C2 and P450 2C1 portions, while the parent proteins
do not carry out this reaction(42, 43) . Whether
substrate access and binding in P450
and
P450
is also influenced by N-terminally residing
amino acids currently remains elusive. However, with our current
findings demonstrating a successful conversion of a
mineralocorticoid-synthesizing enzyme to one that primarily has a
glucocorticoid-synthesizing potential, the paradigm that small changes
in the protein sequence at some critical positions can lead to dramatic
changes in the activity and specificity can be extended to members of
the P450 CYP11 family.