Functional analysis of human cytochrome P450 21A2 variants involved in congenital adrenal hyperplasia

Article abstract of DOI:10.1074/jbc.M117.792465

Cytochrome P450 (P450, CYP) 21A2 is the major steroid 21-hydroxylase, converting progesterone to 11-deoxycorticosterone and 17-hydroxyprogesterone (17-OH-progesterone) to 11-deoxycortisol. More than 100 CYP21A2 variants give rise to congenital adrenal hyperplasia (CAH). We previously reported a structure ofWT human P450 21A2 with bound progesterone and now present a structure bound to the other substrate (17-OH-progesterone).We found that the 17-OH-progesterone- and progesterone-bound complex structures are highly similar, with only some minor differences in surface loop regions. Twelve P450 21A2 variants associated with either saltwasting or nonclassical forms of CAH were expressed, purified, and analyzed. The catalytic activities of these 12 variants ranged from 0.00009% to 30% of WT P450 21A2 and the extent of heme incorporation from 10% to 95% of the WT. Substrate dissociation constants (Ks) for four variants were 37–13,000-fold higher than for WT P450 21A2. Cytochrome b5, which augments several P450 activities, inhibited P450 21A2 activity. Similar to the WT enzyme, high noncompetitive intermolecular kinetic deuterium isotope effects (> 5.5) were observed for all six P450 21A2 variants examined for 21-hydroxylation of 21-d3- progesterone, indicating that C–H bond breaking is a ratelimiting step over a 104 -fold range of catalytic efficiency. Using UV-visible and CD spectroscopy, we found that P450 21A2 thermal stability assessed in bacterial cells and with purified enzymes differed among salt-wasting- and nonclassical-associated variants, but these differences did not correlate with catalytic activity. Our in-depth investigation of CAH-associated P450 21A2 variants reveals critical insigh into the effects of disease-causing mutations on this important enzyme.

Full text of DOI:10.1074/jbc.M117.792465

JBC Papers in Press. Published on May 24, 2017 as Manuscript M117.792465
The latest version is at http://www.jbc.org/cgi/doi/10.1074/jbc.M117.792465
Functional basis of P450 21A2 deficiencies
Functional Analysis of Human Cytochrome P450 21A2 Variants Involved in Congenital Adrenal
S
Hyperplasia*
1
2
Chunxue Wang , Pradeep S. Pallan, Wei Zhang, Li Lei, Francis K. Yoshimoto , Michael R.
3
3
Waterman, Martin Egli , and F. Peter Guengerich
Department of Biochemistry, Vanderbilt University School of Medicine, Nashville,
Tennessee 37232-0146
Running title: Functional basis of P450 21A2 deficiencies
Address correspondence to: Prof. F. Peter Guengerich, Department of Biochemistry, Vanderbilt
University School of Medicine, 638B Robinson Research Building, 2200 Pierce Avenue, Nashville,
Tennessee 37232-0146, Telephone:
f.guengerich@vanderbilt.edu
OR
1
(615) 322-2261; FAX:
1
(615) 343-0704; E-mail:
Prof. Martin Egli, Department of Biochemistry, 868A Robinson Research Building, 2200 Pierce Avenue,
Nashville, Tennessee 37232-0146, Tel.: 615-343-8070; FAX: 615-322-7122; E-mail:
martin.egli@vanderbilt.edu
Keywords: X-ray crystallography, cytochrome P450, steroid biosynthesis, circular dichroism, kinetic
isotope effects, congenital adrenal hyperplasia variants
ABSTRACT
Cytochrome P450 (P450, CYP) 21A2 is
5.5) were observed for all six P450 21A2 variants
examined for 21-hydroxylation of [21-d ]-
3
the major steroid 21-hydroxylase, converting
progesterone to 11-deoxycorticosterone and 17α-
hydroxy(OH)progesterone to 11-deoxycortisol.
More than 100 CYP21A2 variants give rise to
congenital adrenal hyperplasia (CAH). We
previously reported a structure of wild-type (WT)
human P450 21A2 with bound progesterone and
now present a structure bound to the other
substrate (17α-OH progesterone). We found that
the 17α-OH progesterone- and progesterone-
bound complex structures are highly similar, with
only some minor differences in surface loop
regions. Twelve P450 21A2 variants associated
with either salt-wasting (SW) or nonclassical (NC)
forms of CAH were expressed, purified, and
analyzed. The catalytic activities of these 12
variants ranged from 0.00009% to 30% of WT
P450 21A2, and the extent of heme incorporation
from 10% to 95% of the WT. Substrate
progesterone, indicating that C-H bond breaking is
a rate-limiting step over a 10 -fold range of
4
catalytic efficiency. Using UV-visible and CD
spectroscopy, we found that P450 21A2 thermal
stability assessed in bacterial cells and with
purified enzymes differed among SW- and NC-
associated variants, but these differences did not
correlate with catalytic activity. Our in-depth
investigation of CAH-associated P450 21A2
variants reveals critical insight into the effects of
disease-causing mutations on this important
enzyme.
4
Cytochrome P450 (P450 or CYP) 21A2,
an enzyme located in the adrenal cortex, catalyzes
the 21-hydroxylation of both progesterone and
17α-hydroxy (OH) progesterone, forming 11-
deoxycorticosterone
and
11-deoxycortisol,
respectively (2-4). Deficiencies in the CYP21A2
gene are common and are involved in ~ 95% of
the cases of congenital adrenal hyperplasia (CAH)
(2, 3, 5-7), one of the most common inborn errors
of metabolism. The incidence of CAH is ~
1/15,000 worldwide, with well over 100 different
genetic variants having been diagnosed. The
dissociation constants (K
s
) for four variants were
3
7–13,000 fold higher than for WT P450 21A2.
5
Cytochrome b , which augments several P450
activities, inhibited P450 21A2 activity. Similar to
the WT enzyme, high noncompetitive
intermolecular kinetic deuterium isotope effects (≥
1
Copyright 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Functional basis of P450 21A2 deficiencies
phenotypes are generally classified as non-
classical (NC), simple virilizing (SV), and salt-
wasting (SW), in order of increasing severity (2, 3,
29) was replaced by nucleotides coding for the
decameric peptide MAKKTSSKGK, and the C-
terminus was extended by 18 nucleotides encoding
six histidines (9).
6
). The great majority of mutations in CYP21A2
can be traced to the non-functional pseudogene of
P450 21A2 (CYP21A1P), which is found in more
than 70% of all Caucasians. The pseudogene is
We determined the crystal structure of the
complex between P450 21A2 and 17α-OH
progesterone at 3.3 Å resolution. An illustration of
the overall fold of the complex is depicted in Fig.
1A, and selected crystal data, diffraction data
collection, and refinement parameters are listed in
Table 1. Crystals of the complex with 17α-OH
progesterone were isomorphous to those of the
complex with the substrate progesterone and
contain three independent copies in the
asymmetric unit of space group C2. The
conformation of P450 21A2 in complex with 17α-
OH progesterone is very similar to that adopted by
the protein with progesterone bound. The root
mean square deviation for 439 Cα atom pairs in
the overlaid structures is 0.7 Å. The only
conformational difference between the two P450
structures concerns residues 411 to 418 in the
extensive random coil region that links helices L
and M (Fig. 1C). In the higher-resolution (2.64 Å)
complex with progesterone (10), this turn was not
defined in the electron density map. In the
complex with 17α-OH progesterone, all residues
that are part of the turn in the protein chain could
be completely built into the electron density for
the three independent molecules. As expected
from the lower resolution of the structure of the
complex with 17α-OH progesterone, the side
chains of many residues mapping to the surface of
the P450 protein were only partially defined in the
electron density or were missing completely and
were modeled without side chains. Similarly,
inspection of the electron density maps for the
9
8% identical to the functional gene and
crossovers, sequence exchanges, and apparent
gene conversion between functional gene and
pseudogene contribute to frequent variations in the
P450 21A2 protein and the occurrence of CAH (2,
3
, 6, 7).
We published a crystal structure of bovine
P450 21A2 bound with the substrate 17α-OH
progesterone (8), which provided insight into roles
of some of the residues involved in the enzymatic
deficiencies. Using our bovine P450 21A2
structure, New and her associates modeled the
human P450 21A2 enzyme and discussed how the
low activities of some of the variants might be
explained (9). We subsequently crystallized and
published a structure of human P450 21A2 bound
with its other substrate, progesterone (10). This
structure showed some important differences with
the bovine enzyme, and we discussed how these
could effect the conclusions reached with
modeling based on the bovine enzyme (11).
However, even with the P450 21A2 structures
available, it is not clear what all of the factors
causing low catalytic activity are.
We now report a structure of human P450
2
1A2 bound to its substrate 17α-OH progesterone.
In addition, we expressed and purified 12 human
P450 21A2 variants and measured kcat and Km
parameters for 21-hydroxylation of both
progesterone and 17α-OH progesterone. Large
kinetic deuterium isotope effects were observed
with all six of the variants examined, indicating
that C-H bond breaking is a rate-limiting step in
all cases. The stability of the variants was
addressed in several ways. We discuss possible
reasons for low activity of the variants, especially
those variants not analyzed previously (11).
1
7α-OH progesterone complex did not reveal a
second substrate molecule bound at a distal site in
the structure of the progesterone complex (10). A
closer look at the active sites shows that the
orientations and positions of the two substrates are
virtually identical (Fig. 1D). The only noteworthy
deviation concerns the orientation of the keto
group whereby the torsion angles around the C17-
C20 bond differ by about 90° in the progesterone
and 17α-OH progesterone substrates. In the case
of the latter, the C-20 keto oxygen and 17α-
hydroxyl group are pointing in roughly opposite
directions. However, in both the progesterone and
RESULTS
Structure of Wild-type P450 21A2 Bound
to 17α-OH Progesterone— In the open reading
frame of human P450 21A2, the region encoding
the N-terminal transmembrane helix (residues 1–
2

Functional basis of P450 21A2 deficiencies
1
7α-OH progesterone complexes the C21 methyl
analysis, including one NC variant and three SW
variants (Table 4). Wild-type P450 21A2 bound
both substrates (progesterone and 17α-OH
group points into the heme system, with distances
between C21 and Fe of 4.0 Å and 4.3 Å,
respectively (A molecules).
3
+
progesterone) tightly, with K values < 0.1 µM and
s
difficult to estimate accurately using spectral
determinations. Although some of the variants still
bound the substrates with low-µM affinity (Table
Catalytic Efficiencies of Variants—
Twelve clinically observed variants of P450 21A2
were selected for further study, including three
categorized as NC and nine as SW. The UPLC-
UV assays for 21-hydroxylation activity are very
robust because of the high catalytic efficiency of
the wild-type enzyme, and the activity could be
4), the K values clearly showed much less affinity.
s
The difference between the NC variant V282L and
the three SW variants was not obvious. It should
be emphasized that the Ks values do not
necessarily reflect productive binding. The extent
of the decrease in substrate binding is not enough
to explain the attenuated catalytic efficiency
(Table 2, Fig. 2) for the three SW variants (G65E,
G292S, G292C).
6
measured over a 10 -fold range of catalytic
efficiency (Fig. 2). Even the NC variants showed
at least a 2-order of magnitude decrease in
catalytic efficiency, based upon the combined
measured P450 plus cytochrome P420 contents of
the enzymes. The SW variants showed decreases
Lack of Stimulation by Cytochrome b —
5
3
6
of > 10 -fold, up to 10 -fold. It is of interest to
note that (i) all of the changes are the result of
single-amino acid substitutions, (ii) that the amino
acid change at a certain site can have a strong
effect (e.g. P31L vs. P31Q), and (iii) the clinical
classification of the severity of the disease (SW vs.
NC) does not completely match the residual
catalytic activity.
Some P450 activities show remarkable stimulatory
effects of cytochrome b (e.g. P450 17A1 17α,20-
5
lyase activity (13-15)). We examined P450 21A2
for a possible effect, under a variety of conditions
of cytochrome b concentration. Cytochrome b5
5
reduced the activity ~ 4-fold (Fig. 3). The
significance of this attenuation is not clear, but
cytochrome b was clearly not stimulatory and
5
does not appear to be a factor influencing in vivo
activity, in contrast to P450 17A1 (16, 17). In the
way of a caveat, we only analyzed the effect of
Heme Incorporation into P450 21A2
Variants—One issue in the proper folding of P450
2
1A2 variants is the proper incorporation of the
cytochrome b under the typical reconstitution
5
heme prosthetic group. An approximate index of
conditions used with P450 21A2 and in other
systems (13-15) and cannot exclude the possibility
that another system might show different effects.
heme incorporation is provided by the A /A
280
4
17
ratio of the purified variants, in that only one of
the 12 variants examined involved a change in
tryptophan (which would lower A ) (Table 3). All
2
80
Kinetic Deuterium Isotope Effects—Wild-
type human P450 21A2 showed high kinetic
deuterium isotope effects for the 21-hydroxylation
of both progesterone and 17α-OH progesterone,
indicating that C-H bond breaking is one of the
rate-limiting steps in the catalytic cycle (10, 18).
With all six of variants we selected for analysis,
of the mutants showed some decrease in heme
content except G292S, with the level decreased to
1
/10 in several variants (P31Q, W303R, R357W).
The total heme content of the purified
P450 21A2 variants includes both P450 and
cytochrome P420, a term collectively used for
perturbed P450 that is enzymatically inactive (12).
The fraction of the heme in the different forms
varied considerably, from being almost totally
P450 (see Fig. 7A below) to almost entirely
cytochrome P420 (P31Q, G65E, L108R, W303R,
R357W, and R409C; Supplemental Data Fig. S1).
the 21-hydroxylation of [21-d ]-progesterone was
3
considerably slower than progesterone (Fig. 4).
The results are at least qualitatively similar to
those we previously reported with wild-type
human P450 21A2 (10) and implicate C-H bond-
breaking as a rate-limiting step, even though the
4
rates vary 10 -fold.
Substrate Binding to Selected P450 21A2
Variants—Four variants were selected for binding
3

Functional basis of P450 21A2 deficiencies
Assays of Protein Stability—We utilized
two different assays of temperature-dependent
protein stability.
progesterone (10) also revealed a substrate
molecule at a distal site that matches the site of the
second 17α-OH progesterone in the structure of
the complex with bovine 21A2. Most likely owing
to the lower resolution of the structure of the
human 21A2 enzyme with 17α-OH progesterone
One series of studies was done on
temperature-dependence of CD spectra of purified
P450 21A2 variants. The CD spectra of some of
the variants were relatively normal, i.e. similar to
the wild-type protein (Fig. 5). The temperature
dependence of protein integrity was also analyzed,
and the differences between the wild-type and
mutant proteins were found to be rather limited
(
this work, Fig. 1), the electron density at this
distal site was not defined well enough to identify
a second substrate molecule.
The availability of structural, activity,
stability, and substrate binding data offers an
(
Supplemental Data Fig. S1). Two of the SW
opportunity to potentially gain
a
refined
mutants (G292C and L108R) showed very
distorted CD spectra but so did the NC-variant
V282L.
We also used an alternative approach that
utilized the variant P450s in the Escherichia coli
cells, without purification (21) (which might have
understanding of the phenotypes of diverse CAH
variants and the different degrees to which SW,
SV, and NC mutations affect the P450 21A2
enzyme. Among the variants we investigated in
this work are rare mutations as well as common
pseudogene-derived ones. The latter include
I172N (SV), P31L (NC), and V282L (the most
common NC variant; Richard J. Auchus, personal
communication).
induced
denaturation).
Reduced
P450-CO
complex spectra of P450 variants were acquired in
bacterial cells following 10-min exposure to
increasing temperatures (Fig. 6). The presence of
the 450 nm band is indicative of structurally-intact
protein, and the band at 420 nm is indicative of
Ile-172 (I172N variant, SV) is located in
the middle of helix E that is part of helical bundle
(
C, E, I, and M helices, Fig. 1A). Ile-172 is part of
cytochrome P420,
a
denatured form (12).
a relatively hydrophobic environment including
Val-140, Leu-176, and Leu-434 and also a
hydrophilic residue, Glu-438 (Fig. 7A). Replacing
isoleucine with asparagine affects these
hydrophobic interactions to some extent but also
allows H-bonds between the amino group of Asn-
Interestingly, some of the variants with very low
catalytic efficiency (Table 2, Fig. 2) expressed
reasonably well, as judged by the spectra. All
P450 21A2 variants were denatured to some extent.
Although some variants had very low catalytic
efficiency (e.g. G292C), they had thermal
stabilities similar to wild-type P450 21A2, as
judged by the temperatures at which one-half the
1
72 and Glu-438 (Oε2; 3.06 Å) and between the
side chain carbonyl of Asn-172 and the main chain
N-H of Val-140 (3.60 Å). I172N displayed a
considerable loss in catalytic activity (~2%
residual activity with 17α-OH progesterone, Table
4
50-nm absorbance spectra were lost. In these
assays, the spectra seen with P31Q, G65E, L108R,
W303R, R357W, and R409C (Supplemental Data
Fig. S2) were not strong enough at 450 nm to
apply the assays, reflecting the low intrinsic ability
to properly incorporate the heme prosthetic group.
2
) and a 7-fold reduction in heme incorporation
compared with wild-type P450 21A2 (Table 3).
Val-282 (V282L variant, NC) is located
near the N-terminal end of the long I-helix and
surrounded by hydrophobic residues from the G-
DISCUSSION
(
Leu-242) and H-helices (Met-258, Met-261, and
The first structure of P450 21A2 was that
Leu-262) (Fig. 7B). The increased size of the
leucine relative to the valine side chain can be
accommodated to some degree at this site,
consistent with the residual activity and the NC
phenotype (Table 2). The activity we measured
here is considerably lower than that reported
earlier (20% for progesterone and 50% for 17α-
of
a
slightly modified bovine enzyme
(
T241R/L442A) with the substrate 17α-OH
progesterone (8), and two molecules of this
substrate were present, one bound at the active site
and the other at a distal site adjacent to the Fʹ helix.
We have not obtained a structure of the bovine
enzyme with the substrate progesterone bound.
The structures of human P450 21A2 bound with
OH
progesterone;
(22).
http://www.cypalleles.ki.se/cyp21.htm)
4

Functional basis of P450 21A2 deficiencies
Among the variants for which we evaluated the
stability change of the Fe -CO spectrum based on
heating, the V282L mutant showed the greatest
Methylene groups of the arginine side chain are
surrounded by relatively hydrophobic side chains,
including Trp-303, Pro-464, and Leu-472, whereas
the guanidino moiety faces Asp-44, Gln-390, and
Gln-463. The mutation to tryptophan results in a
stacking interaction between the side chains of
Trp-357 and Trp-303 (ca. 3.8 Å separation, Fig.
7E). The variant displayed some residual catalytic
activity (<1% with progesterone or 17α-OH
progesterone, Table 2) (but displayed the lowest
level of heme incorporation among the
investigated CAH variants, <10% relative to wild-
type P450 21A2, Table 3).
2
+
reduction in T relative to the wild-type protein
m
(
Fig. 6E). This variant also displayed
significantly distorted CD spectrum even at 25 °C
Fig. 5D), supporting the notion that the even the
a
(
addition of a single methyl group of the leucine
side chain relative to valine is not well tolerated.
This variant also showed very poor affinity for
substrates (Table 4).
Gly-292 (G292C variant, SW) maps to the
central region of the I-helix and lies in close
proximity of substrate and heme (Fig. 8C). The
cysteine mutation results in steric clashes with ring
A of 17α-OH progesterone and progesterone and
potentially heme. The CD spectrum was very
distorted even at 25 °C (Fig. 5B). The complete
loss of activity in this severe mutant is therefore
Arg-409 (R409C variant, SW) is located
in a highly polar environment in the loop region
that connects helices L and M and is part of the
highly conserved EXXR motif (see Fig. 4D in
(11)). The mutation to cysteine introduces a
hydrophobic and less sterically demanding entity
into this polar environment (Fig. 7F). Thus, the
cysteine side chain is unlikely to create any short
contacts and it appears that it can be
accommodated between the side chains of Trp-406
and Asn-416. Given the intricate interactions that
Arg-409 is engaged in (H-bonding with four
neighboring residues and involving both side and
main chain atoms of these, Fig. 7F), none of which
can be mimicked by cysteine, it is perhaps
surprising that the R409C CAH variant still
displays between ca. 0.5 and 2.5% of the wild-type
activity (Table 2).
not surprising (Table 2), despite
incorporation that was only marginally affected
75% of wild-type enzyme, Table 3). The effect of
a
heme
(
temperature was not great, surprisingly (Figs. 5, 6,
Supplemental Data Fig. S1). Binding of of
progesterone was very poor (Table 4) but 17α-OH
progesterone bound reasonably well. Interestingly,
progesterone binding (relative K ) is dramatically
S
reduced compared to wild-type 21A2 (more than
4
1
0 -fold, Table 4), although the effect is less
prominent in the case of 17α-OH progesterone.
The G292S variant (SW) is less damaging
We had evaluated potential steric and
than the cysteine mutant as indicated by activity
data that show ~100-fold reduction compared to
wild-type P450 21A2 (Table 2). The serine side
chain is slightly smaller and the γ-hydroxyl may
be more easily accommodated given its vicinity to
more polar moieties such as the 17α-OH group
and the main chain carbonyl groups of Asp-288
and Leu-289 (Fig. 7D). Indeed, this variant binds
progesterone about 25-fold more tightly than the
P450 G292C variant (Table 4). Neither the G292C
nor the G292S variant exhibits a dramatically
electrostatic effects of 24 SW, SV, and NC CAH
variants based on the previously determined
crystal structure of the complex between human
P450 21A2 and progesterone (10, 11). Among
them were the P31L (NC), P31Q (SW), G65E
(
(
SW), L108R (SW), T296N (SW), and W303R
SW) variants, for which we now report catalytic
activities with the progesterone and 17α-OH
progesterone substrates (Table 2). Consistent with
the severity of the CAH phenotype, the P31L
mutant does not hamper catalysis to the same
extent as the P31Q mutant (the latter is basically
inactive). The deviating magnitude of the activity
reductions seen for the two variants are matched
by those on heme incorporation (3-fold lower for
P31Q, Table 3), and the loss in stability at least for
P31L relative to the wild-type protein is quite
minor (Fig. 6B). Pro-31, along with an adjacent
2
+
altered thermal stability. In fact, in the Fe -CO
spectra the T of the latter was only 2 °C below
m
that of wild-type P450 21A2 (Fig. 6D). However,
just like the V282L variant, G292C showed a
significantly changed CD spectrum relative to
wild-type protein even at 25 ° C (Fig. 5B).
The Arg-357 (R357W) variant (SW) is
located at the C-terminus of the K helix.
5

Functional basis of P450 21A2 deficiencies
proline, marks the site of a sharp turn in the
direction of the protein chain and help anchor the
N-terminal hydrophobic tail that serves the
attachment of P450 21A2 to the membrane. The
leucine mutation is sterically compromising but
less severe, from a structural perspective, than the
glutamine mutation that affects both sterics and
electrostatics/H-bonding in comparison to proline.
Gly-65 (G65E variant, SW) is close to the
surface and at the apex of the short loop that
connects the β2 and β3 strands, facing the N-
terminal residue of the Fʹ helix (Pro-214, Fig. 1A).
Mutation to glutamate introduces a polar residue
into a patch of hydrophobic side chains from
this mutation to heme, the fraction of heme
measured relative to wild-type 21A2 still amounts
to 40% (Table 3).
In comparison to the above L108R variant,
mutation of Thr-296 to asparagine—although
classified as an SW CAH phenotype as well—
affects the catalytic activity to a smaller degree:
0.05% and 4% of wild-type 21A2 activity with the
progesterone and 17α-OH progesterone substrates,
respectively (Table 2). Heme incorporation was
also increased relative to the L108R variant (62%
and 25%, respectively, Table 3) and the thermal
stability was virtually the same as for wild-type
protein (Fig. 6F, -1 °C). Thr-296 is located in the
residues Leu-38, Leu-40, Leu-64, Leu-66, Val-212, long I helix and sits above the side of the heme
3
+
and Ile-213. In addition to disturbing the apolar
character of this location, the relatively long
glutamate side chain creates steric challenges, and
the significant loss of catalytic activity with this
variant (ca. 0.1% of wild-type protein with both
substrates, Table 2) is in line with the anticipated
structural consequences. The poor incorporation of
heme (25% of wild-type 21A2, Table 3) and the
significantly reduced binding particularly of
progesterone (some 2,000-fold relative to wild-
type, Table 4) are perhaps more surprising, given
the location of Gly-65 close to the surface and at a
considerable distance from active site and heme.
Leu-108 (L108R variant, SW) is located
(5.2 Å distance between Oγ and Fe ) and in the
vicinity of ring A of the substrate molecule (5.5 Å
distance between Oγ and O17) (Fig. 1D). In the
modeled Asn-296 mutant, the above distances
3
+
would be reduced to 4.7 Å and 3.1 Å (Nδ2…Fe
and Nδ2…O17, respectively). Apparently
swapping threonine and asparagine does not
entirely abolish activity (some 25-fold reduced
compared to wild-type protein with 17α-OH
progesterone), an observation that is noteworthy
given how closely it occurs to substrate and heme
and given the more drastic consequences of
mutations farther away from the active site (e.g.,
P31Q and L65R).
in a loop that precedes helix C (Fig. 1A) and
positioned immediately adjacent to methyl and
propionic acid substituents of heme. Introduction
of the longer arginine with its positively charged
guanidino moiety in place of leucine that sits in a
partly hydrophobic region, but also has His-120,
Lys-121, and Asp-288 among its neighbors was
expected to distort the conformational/electronic
balance. Consistent with this assessment the CD
spectrum for the L108R variant displays
significant changes relative to wild-type protein
even at 25 °C (Fig 5H). Moreover, the complete
loss of activity (Table 2) is in line with the
structural picture and the severity of the CAH
phenotype. The activities based on in vitro kinetic
assays and using purified mutant protein reported
here are lower than those reported earlier and
based on experiments conducted with transiently
transfected COS-1 cells (ca. 0.3% activity with
both substrates relative to wild-type protein (23)).
Perhaps surprisingly, given the close vicinity of
The consequences of the T296N mutation
are also more limited than those seen for another
SW CAH variant, W303R, Trp-303 being located
farther along the I helix and thus more removed
from heme and substrate than Thr-296. Mutation
of Trp-303 to arginine destroys the catalytic
activity with both substrates (Table 2) and also
affects heme incorporation to a very significant
extent (11%, Table 3). An inspection of the
structures of the human P450 21A2 substrate
complexes suggests that an arginine at position
3
03 is likely to create a repulsion with Arg-357
that sits at the end of the K helix (Fig. 1A). In the
model of the mutant, the guanidino moieties of the
two arginines are positioned at 3.5 Å from one
another. Interestingly, the R357W mutation is also
classified as an SW CAH variant, whereby its
activity is slightly higher than that of W303R (ca.
.01% and 0.1% for progesterone and 17α-OH
progesterone, respectively, Table 2). Moreover,
the levels of heme incorporation were quite similar
0
6

Functional basis of P450 21A2 deficiencies
for the two variants, ~ 10% (Table 3). The most
likely reasons for the detrimental effects on
activity and heme incorporation of these two
mutations are altered positions and mobility of the
I helix that spans the entire core of the protein and
runs by the heme/active site. These changes in
conformation and dynamic behavior occur either
as a consequence of replacement of the Trp(303)-
Arg(357) pair by Arg-Arg (W303R variant) or
Trp-Trp (R357W variant) pairs (in the model of
the latter, the two indole moieties are ideally
stacked at 3.4 Å).
(Supplemental Data Fig. S1). Overall, the CD
results by themselves did not provide explanations
for the defective nature of the variants.
2
+
The Fe ·CO results clearly showed less
stability of all variants (Fig. 6 and Supplemental
Data Fig. S2). Interestingly, the temperature
dependence of these was rather invariant, i.e. they
started with a certain fraction of cytochrome P420
but the degree to which the residual P450 broke
down was similar (V282L was somewhat faster,
Fig. 6E).
Overall, we can conclude that a variety of
issues are associated with the different clinical
loss-of-function variants. The single amino acid
substitutions can produce dramatic losses of
functional integrity (Fig. 5), ability to bind the
heme prosthetic group (Table 3, Fig. 6,
Supplemental Fig. S2), and ability to bind
substrates (Table 4). Collectively these problems
result in low catalytic efficiency (Fig. 1, Table 2).
Nevertheless, C-H bond breaking appears to be the
rate-limiting step across several orders of
magnitude of catalytic efficiency (Figs. 1 and 4,
Table 2).
Overall, we find that single amino acid
substitutions can have remarkable effects. All of
the variants studied here were identified in clinical
practice (11). The summaries of catalytic activities
of
P450
21A2
activities
(11)
(
http://www.cypalleles.ki.se/cyp21.htm) are rather
crude and contain values measured with many
different expression systems and cellular and other
assays. We provide here (Table 2, Fig. 1) and
elsewhere (11) a robust set of k , K , and k /K
m
cat
m
cat
values obtained under precise conditions with
UPLC-UV measurements.
Although it is popular to use
The ability of small changes in structure to
influence large changes in catalytic efficiency
seems remarkable, but it is of use to consider the
Eyring equation
crystallographic results to consider alterations of
substrate binding to explain properties of variants,
our results suggest that the losses of catalytic
activity (Table 2) are too large to be explained by
attenuation of substrate binding (Type I) (24)
although the heme perturbation assay may not
necessarily reflect productive substrate binding.
Overall, the changes of single amino acids often
have dramatic effects on structural properties of
P450 21A2. Evidence for this is seen in the
incorporation of heme, both total heme (Table 3)
and the content of P450 vs. inactive cytochrome
P420 (Fig. 6, Supplemental Data Fig. S1). Some of
the loss of heme from certain mutants might have
occurred during purification, but large differences
were also seen in the bacterial cells (Fig. 6,
Supplemental Data Fig. S2).
∆!±
ꢀ ꢀ
!
!
ꢀ!"#
=
ꢀ
!"
ℎ
(kB is the Boltzman constant, h is Planck’s
constant) (25), and a mutation yielding a ΔΔG of
-
1
1
.3 kcal mol leads to a 10-fold change in rate,
-1
and a ΔΔG of 6.4 kcal mol leads to a 50,000-fold
change in kobs.
In an earlier study of P450 21A2 we had
mapped many SW, SV, and NC mutations onto the
3
D structure of the enzyme and found patterns of
distribution characteristic of the three types of
CAH variants (11). Thus, SW-causing mutations
that are more common than SV variants were
spread throughout the protein, often involving
hydrophobic amino acids and dotting the active
site area and heme-binding pocket. By comparison,
SV- and NC-causing mutations concern regions
farther removed from the center and, particularly
in the case of the NC variants, commonly map to
areas on or near the enzyme surface, where they
could be expected to be less damaging to stability
The CD spectra of some of the purified
variants indicated considerable distortion, e.g.
G292C, V282L, and L108R (Fig. 5). However,
other variants with catalytic efficiencies just as
low did not show the losses of α-helicity (e.g.,
W303R). The loss of α-helicity with increasing
temperature was no worse for the defective
mutants than for the wild-type enzyme
7

Functional basis of P450 21A2 deficiencies
and activity. The comparison of activities of SW,
SV, and NC variants, based on various assays,
confirmed the greater loss of activity in enzymes
exhibiting the SW phenotype relative to those
classified as SV and particularly NC (11). The
detailed analysis of a dozen variants presented
here and—using structural, activity, folding
stability and spectroscopic assays to measure heme
incorporation—demonstrates in some cases that
steady-state kinetic assays using a purified P450
construct was extended by 18 nucleotides
encoding a (His) tag. The synthesized cDNA was
6
inserted into the pET17b expression vector (EMD
Millipore, Billerica, MA) and the plasmid co-
transformed with chaperone pGro12 plasmid into
E. coli BL21-Gold DE(3) competent cells.
Following expression the protein was purified with
nickel affinity chromatography and then used for
further chromatography in the absence of
detergent with an SP Fast-Flow Sepharose column
(GE Health Products). The same basic methods
were used for the P450 21A2 variants.
2
1A2 variant and cell-based measurements of
activity can produce considerably different
outcomes. Thus, the most common NC variant,
V282L, displayed much lower activity in the
kinetic assays than in earlier cell-based
experiments. Not only did this mutation impact
activity to a considerable degree, but it also
showed poor substrate binding and the greatest
loss in thermodynamic stability among all those
CAH variants tested. Similarly, mutations at the
surface can be equally or more damaging than
those at the heart of the enzyme as demonstrated
by the P31Q (SW) and the G292S/G292C (SW)
variants, respectively. The crystal structure
provides some insight into the origins of the
relative degree of damage caused by individual
mutations. Overall, our in-depth investigation of
selected types of CAH variants and relying on an
array of experimental approaches provides new
insight into disease-causing mutations in the P450
Purification of Other Enzymes—Rat
NADPH-P450 reductase (26) and human
cytochrome b
purified as described elsewhere.
(27) were expressed in E. coli and
5
Assays of Catalytic Activity—The 21-
hydroxylation of progesterone and 17α-OH
progesterone were assayed as described in detail
elsewhere (8, 10). P450 concentrations were
adjusted accordingly to avoid >20% disappearance
of substrate during incubations. The products were
separated by UPLC, with detection at 240 nm
using a photodiode array system. Rates of product
formation were plotted vs. substrate concentration
to obtain hyperbolic Michaelis-Menten plots,
which were subjected to non-linear regression in
GraphPad Prism to estimate k and K values.
cat
m
2
1A2 enzyme which is among the metabolic
enzymes with the largest number of naturally
occurring variations (>>100) that alter activity.
Spectroscopy—UV-visible spectra were
recorded with an Aminco-OLIS DW2a
spectrophotometer. Substrate binding assays were
EXPERIMENTAL PROCEDURES
done
spectrophotometrically
as
described
Chemicals—Progesterone and 17α-OH
progesterone were purchased from Sigma Aldrich
or Steraloids (Witton, NH) and used without
elsewhere (10, 24).
Circular dichroism (CD) spectroscopy was
performed with an Aviv spectrophotometer
(Model 215, Aviv Biomedical, Lakewood, NJ).
The instrument was pre-equilibrated at 25 °C for
further purification. [21-d ]-Progesterone was
3
synthesized as described elsewhere (18).
1
0 min prior to the acquisition of data. The
Expression and Purification of Human
P450 21A2—Recombinant P450 21A2 enzyme
encompassing amino acids 30-495 was expressed
and purified as described in (10). Briefly, in the
CYP21A2 open reading frame the region coding
for the 29-residue long N-terminal trans-
membrane helix (residues 1–29) was replaced by
nucleotides encoding the P450 2C3 N-terminal
peptide MAKKTSSKGK. At the 3´-end the
proteins were diluted to total protein
a
concentration of 2 µM with 50 mM potassium
phosphate (pH 7.4) prior to analysis. Each sample
was loaded into a 0.2-cm pathlength quartz cuvette
and allowed to equilibrate inside the cell of CD
spectrophotometer for 5 min at 25 °C. Spectra
were recorded from 25 to 85 °C, with increments
of 10 °C. Each scan was collected from 185 to 300
nm in the continuous scanning mode at each
-
1
temperature, with a scan speed of 100 nm min
8

Functional basis of P450 21A2 deficiencies
and a 1 nm bandwidth. CD spectral data were
smoothed, buffer-subtracted, and corrected to
molar ellipticity on the basis of protein
concentration and molecular weight. Calculations
of α-helix content were made using the formulae
of Greenfield and Fasman (19) as modified by
Chen and Yang (20).
Larger crystals of the complex grew from micro-
seeded droplets in about a week. Crystals were
mounted in nylon loops, swiped through a droplet
of 25% glycerol (v/v) in mother liquor, and flash
frozen in liquid nitrogen.
X-Ray
Data
Collection,
Structure
The experiments in which P450-CO
Determination, and Refinement—Diffraction data
were collected at 100 K on the insertion device
beam line (21-ID-G) of the Life Sciences
Collaborative Access Team (LS-CAT), located at
Sector 21 of the Advanced Photon Source,
Argonne National Laboratory (Argonne, IL), using
a wavelength of 0.97857 Å and a Mar300 CCD
detector. Data were processed with the program
HKL2000 (28) and selected crystal data and
refinement statistics are summarized in Table 1.
The structure of the P450 21A2-17α-OH
progesterone complex was determined by the
molecular replacement technique with the program
Phaser (29) using the crystallographic coordinates
of the human P450 21A2-progesterone complex as
the search model (10) (PDB ID 4Y8W).
Refinement was carried out with the program
Refmac5 using the TLS option (29, 30). Manual
rebuilding of the model was performed in Coot
(31). The r.m.s.d. values from the superimposition
were calculated with the Superpose routine in
CCP4 (29). All structural figures were generated
with the program UCSF-Chimera (32).
spectra were recorded in bacterial cells were done
as described in detail by Johnston and Gillam (21).
Bacterial cells were treated with sodium dithionite
to establish baseline absorbance, and the CO was
then added. The cells were allowed to equilibrate
for 10 min at each temperature before
measurements. The 450 nm absorbance data were
fit to sigmoidal plots in Prism software (GraphPad,
La
Jolla,
CA)
(Y=Bottom
+
(Top-
Bottom)/(1+10^((LogEC50-X))).
Crystallization of the P450 21A2-
Substrate Complex—The human P450 21A2-17α-
OH progesterone complex was formed by adding
substrate (dissolved in ethanol) to P450 21A2 in a
:5 (enzyme:substrate) ratio, followed by mixing
and concentration to 25 mg protein/ml in 50 mM
potassium phosphate buffer (pH 7.2) containing
1
5
% glycerol (v/v), 0.1 mM DTT, 0.1 mM EDTA,
and 100 mM NaCl. Aliquots of the complex
solution were frozen in liquid N and then stored at
2
-
80 °C. Complex crystals were grown using the
sitting-drop vapor diffusion technique and mixing
equal volumes (200 nl) of complex solution and
mother liquor (0.20 M tri-ammonium citrate (pH
Modeling of Selected Variants in the
Structure of the P450 21A2-17α-OH Progesterone
Complex—For visualization of SW, SV, and NC
CAH-causing mutations in the 3-dimensional
structure of the P450 21A2 complex, we used
UCSF-Chimera to swap residues and generate
illustrations (32). The residue numbers in the text
and illustrations refer to proteins encoded by the
human 21-hydroxylase gene with GenBank
number M26856.1 (wild-type [wt] protein) or the
corresponding genes with mutations and are
consistent with the numbering used in previous
work from our group (10, 11).
7
.0) and 20% (w/v) polyethylene glycol 3350),
and equilibrating droplets against 60 µl of
reservoir solution at 20 °C. Microcrystals were
obtained within two weeks and, in order to
improve their size, one round of micro-seeding
was performed. Wells containing microcrystals
were washed with 10 µl of reservoir buffer and
transferred to a microcentrifuge tube with 40 µl of
mother liquor. After vortex mixing the seeds for
two minutes and diluting 1:1000 with mother
liquor, 200 nl of the seed solution was mixed with
2
00 nl of protein-substrate complex for each drop.
Acknowledgments: We thank L. D. Nagy and T. T. N. Phan for purifying NADPH-P450 reductase, E.
M. J. Gillam for suggesting the bacterial cell P450-CO binding experiments and technical advice for
doing the assays, D. W. Wright for the use of his CD spectrophotometer, A. L. Bitting for her technical
help with CD experiments, and K. Trisler for her assistance in preparation of the manuscript.
9

Functional basis of P450 21A2 deficiencies
Conflict of interest: The authors declare that they have no conflicts of interest with the contents of this
article.
Author contributions: C.W. performed the site-directed mutagenesis, purified the variants, ran the
catalytic assays, and collected some of the stability data. P.S.P. solved the structure and initiated the CD
work. W.Z. acquired the CD spectra. L.L. made the wild-type 21A2 expression vector, purified and
crystallized proteins. F.K.Y. synthesized d -progesterone. M.R.W., F.P.G., and M.E. conceived and
3
directed the studies and analyzed the results. F.P.G. and M.E. wrote the manuscript. All authors agree
with the conclusions.
1
0

Functional basis of P450 21A2 deficiencies
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2

Functional basis of P450 21A2 deficiencies
FOOTNOTES
1
Current address: Allergy/Pulmonary and Critical Care Medicine, Vanderbilt University Medical Center,
TN 37232
2
Current address: Dept. Chemistry, University of Texas at Antonio, San Antonio, TX 78249
3
Address correspondence to either of these authors.
S
The on-line version of this article (available at http://www.jbc.org) contains Supplemental Data.
4
The abbreviations used are: CAH, congenital adrenal hyperplasia; NC, non-classical; OH, hydroxy, P450
D
H
D
(
or CYP), cytochrome P450; SV, simple-virilizing; SW, salt-wasting. The conventions V = k / k and
cat
cat
D
H
H
D
D
(
V/K) = ( k / K )/( k / K ) of Northrop (1) are used with kinetic isotope effects.
cat m cat m
*
This work was supported, in whole or in part, by National Institutes of Health Grant R01 GM103937 (to
M.E. and F.P.G.). The content is solely the responsibility of the authors and does not necessarily represent
the official views of the National Institutes of Health.
1
3

Functional basis of P450 21A2 deficiencies
Table 1. Selected crystal data, data collection and refinement parameters for the P450 21A2-17α-
OH progesterone complex.
Data collection
Wavelength [Å]
Space group
0.97857
C2
a
Resolution (outer shell) [Å]
Unit cell constants a, b, c [Å]; β [°]
a, b, c [Å]; α, β, γ [°]
Unique reflections
Completeness [%]
I/σ(I)
43.27 - 3.31 (3.42 - 3.31)
152.43, 88.39, 111.42; 102.1
19,819 (1,982)
92.3 (93.2)
11.8 (1.5)
R-pim
0.060 (0.456)
4.5 (4.4)
Redundancy
Refinement
Phasing method
R-work
Molecular Replacement
b
0.1899 (0.300)
b
R-free
0.2381 (0.296)
c
Number of amino acids
Number of protein atoms
Number of heme/ligand atoms
1,325
10,589
129 / 69
92.5
2
Average B-factor, protein atoms [Å ]
2
Average B-factor, ligand/heme atoms [Å ]
83.8 / 95.8
91.2
2
Wilson B-factor [Å ]
1
4

Functional basis of P450 21A2 deficiencies
R.m.s.d. bond lengths [Å]
R.m.s.d. bond angles [°]
Ramachandran plot: No of favored / allowed /
outlier residues
0.015
2.2
1
,263 / 29 / 15
PDB ID code
5VBU
a
Numbers in parentheses refer to the outer shell.
Outer shell for refinement is 3.40-3.31 Å.
b
c
For three independent molecules per asymmetric unit. Molecules A and C: residues 29−266 /
75−325 / 333−485. Molecule B: residues 29−266 / 275−32 5/ 333−484.
2
1
5

Functional basis of P450 21A2 deficiencies
Table 2. Catalytic activities of 21A2 variants.
-
1
Variant Clinical
phenotype
Substrate
kcat, min
K , µM
m
k /K ,
µM min
Catalytic
efficiency,
cat
m
-
1
-1
%
wild-type
Progesterone 170 ± 4
7α-OH
240 ± 5
Progesterone
Progesterone 23 ± 2
7α-OH
84 ± 7
Progesterone
Progesterone 0.0076 ± 0.0005
0.21 ± 0.03
1.5 ± 0.1
800 ± 120
160 ± 10
100
100
Wild-
type
1
1.2 ± 3.1
19 ± 5
49 ± 7
2.4
30
P31L
P31Q
G65E
L108R
NC
SW
SW
SW
1
1.7 ± 0.58
9.6 ± 1.7
7.3 ± 1.1
0.00080
0.0001
0.0011
±
0.0002
1
7α-OH
0.013 ± 0.001
0.0018
± 0.0003
Progesterone
Progesterone 3.6 ± 0.2
7α-OH
1.9 ± 0.2
Progesterone
Progesterone 0.0059 ± 0.001
5.5 ± 0.8
13 ± 2
0.67 ± 0.10
0.14 ± 0.02
0.082
0.089
1
2.7 ± 0.2
1.7 ± 0.5
0.0022
0.00027
0.00072
±
0.0002
1
7α-OH
0.002 ± 0.0002
0.0012
Progesterone
± 0.0007
Progesterone 0.087 ± 0.002
7α-OH
0.18 ± 0.01
Progesterone
Progesterone 62 ± 3
7α-OH
2 ± 4
1.8 ± 1.0
4.9 ± 0.5
0.049 ± 0.027 0.0061
0.037 ± 0.004 0.023
I172N
SV
NC
1
3.8 ± 0.6
14 ± 2
16 ± 4
2.0
2.3
V282L
1
3.7 ± 0.6
Progesterone
Progesterone 0.046 ± 0.004
12 ± 3
0.0038
0.00047
0.00047
±
0.0009
G292C
SW
1
7α-OH
0.0026 ± 0.0002
3.6 ± 0.7
0.0073
Progesterone
± 0.0001
Progesterone 12 ± 1
7α-OH
3.8 ± 0.3
4.7 ± 0.3
4.6 ± 0.7
2.6 ± 0.2
0.32
0.51
G292S
T296N
SW
SW
1
0.82 ± 0.14
Progesterone
Progesterone 0.46 ± 0.16
7α-OH
1.7 ± 0.3
Progesterone
1.1 ± 0.2
0.42 ± 0.16
6.0 ± 2.7
0.052
3.7
1
0.46 ± 0.2
1
6

Functional basis of P450 21A2 deficiencies
Progesterone 0.0079 ± 0.0005
11 ± 2
2.4 ± 0.5
22 ± 4
22 ± 3
0.00072
0.00014
0.00009
0.0006
0.0091
0.075
±
W303R
SW
1
7α-OH
0.0023 ± 0.0002
0.00095
± 0.00021
Progesterone
Progesterone 0.016 ± 0.001
0.00073
±
0.00014
R357W
R409C
SW
SW
1
7α-OH
0.027 ± 0.002
0.0012
Progesterone
± 0.00015
Progesterone 1.2 ± 0.1
7α-OH
1.8 ± 0.1
Progesterone
0.39 ± 0.08
0.44 ± 0.09
3.0 ± 0.6
4.0± 0.9
0.37
2.5
1
1
7

Functional basis of P450 21A2 deficiencies
Table 3. Heme incorporation into P450 21A2 variants
Variant
Clinical
A /A
280
Fraction relative to
4
17
wild-type
phenotype
Wild-type
0.63
1.0
P31L
NC
0.24
0.07
0.16
0.23
0.09
0.35
0.60
0.47
0.39
0.07
0.06
0.08
0.38
0.11
0.25
0.37
0.14
0.56
0.95
0.75
0.62
0.11
0.095
0.13
P31Q
SW
SW
SW
SV
G65E
L108R
I172N
V282L
G292S
G292C
T296N
W303R
R357W
R409C
NC
SW
SW
SV/SW
SW
SW
SW
1
8

Functional basis of P450 21A2 deficiencies
Table 4. Substrate binding by some 21A2 variants.
Variant
Clinical
Substrate
K , µM
Change compared
to wild-type
s
phenotype
Wild-type
—
Progesterone
0.010 ± 0.003
0.030 ± 0.003
1
1
1
7α-OH Progesterone
Progesterone
7α-OH Progesterone
Progesterone
7α-OH Progesterone
Progesterone
7α-OH Progesterone
Progesterone
7α-OH Progesterone
a
G65E
SW
NC
SW
SW
22 ± 3
2200
83
1
2.5 ± 0.7
V282L
G292C
G292S
16 ± 2
1600
210
1
6.2 ± 1.7
a
130 ± 40
13,000
37
1
1.1 ± 0.4
5.4 ± 0.4
5.0 ± 0.6
540
170
1
a
Subject to more error due to linearity of plots.
1
9

Functional basis of P450 21A2 deficiencies
FIGURES AND FIGURE LEGENDS
FIGURE 1.
Crystal structure of human P450 21A2 in complex with the substrate 17α-OH
progesterone. A, overall view of the complex colored in rainbow style (from N-terminus, blue, to C-
terminus, red) with individual α-helices and β-strands labeled and every tenth residue numbered. Carbon,
3
+
nitrogen, and oxygen atoms of the heme moiety are colored in gray, blue, and red, respectively, and Fe
is shown as an orange sphere. Carbon and oxygen atoms of 17α-OH-progesterone are colored in salmon
and red, respectively. B, quality of the final omit 2Fo-Fc Fourier electron density (1.2σ level) around the
substrate. C, overall view of the superimposed progesterone (pink ribbon; molecule A) and 17α-OH
progesterone complexes (green ribbon; molecule A). Conformational differences between the two
complexes in a portion (residues 411-418) of the long loop connecting helices L and M are indicated with
an arrow. D, close-up view of the active sites of the two superimposed complexes with progesterone and
1
7α-OH progesterone bound. Carbon atoms of substrates and heme are colored in gray and light green,
respectively.
2
0

Functional basis of P450 21A2 deficiencies
1
000
00
Progesterone
NC
1
NC
1
7α-OH progesterone
SW
SW
1
0
1
SW
SW
SV
0
.1
.01
.001
.0001
0
SW
SW
SW
SW SW
0
0
WT
P31 LP 31QG65E
I172N
L108R V282 GL 292 GS 292 CT 296N
W303 RR 357W
R409C
Variant
FIGURE 2.
Catalytic efficiencies of wild-type P450 21A2 and some variants. WT: wild-type.
500
400
300
200
100
0
-
b5
+
b5
0
10
20
30
40
50
[
Progesterone], μM
FIGURE 3.
Inhibition of P450 21A2 by cytochrome b . Progesterone 21-hydroxylation was
5
measured in a reconstituted system containing 2 nM P450 21A2 and 0.60 µM NADPH-P450 reductase in
the absence (!) or presence (") of 0.6 µM cytochrome b . In the absence of cytochrome b , k was 440
5
5
cat
-
1
-1
min and the K was l.7 µM under these conditions. With cytochrome b present, the k was 120 min
m
5
cat
and K was 1.3 µM. Each point on the graph is a mean of duplicate determinations.
m
2
1

Functional basis of P450 21A2 deficiencies
1
5
0
5
0
600
400
200
0
V282L
G65E
A
C
E
B
d0
1
d0
d3
d3
0
5
10
15
20
0
5
10
15
20
20
40
0
0
0
0
0
0
.05
.04
.03
.02
.01
.00
15
G292C
G292S
d0
D
F
d0
1
0
5
0
d3
d3
0
5
10
15
20
0
5
10
15
3
2
1
0
1
1
0
.5
.0
.5
0
T296N
R409C
d0
d0
d3
d3
0
10
20
30
0
10
20
30
40
[Progesterone], µM
FIGURE 4.
Kinetic isotope effects for several variants of P450 21A2. The results of assays with d0-
and [21-d ]-progesterone are shown. From the k and k /K values (estimated using Graphpad Prism),
3
cat
cat
m
D
D
D
D
D
the following estimates of V and (V/K) were calculated: A (G65E), V 17, (V/K) 13; B (V282L), V 31,
D
D
D
D
D
D
D
(
V/K) 7.8; C (G292C), V 8.7, (V/K) 5.5; D (G292S), V 12, (V/K) 7.7; E (T296N) V 5.1, (V/K) 12; F
D
D
(
R409C), V 10, (V/K) 5.5.
2
2

Functional basis of P450 21A2 deficiencies
Fig. 5
A
C
E
G
B
wild-type
G292C
D
V282L
G292S
F
P31L
R409C
H
W303R
L108R
Wavelength, nm
2
3

Functional basis of P450 21A2 deficiencies
FIGURE 5. Thermal stability of P450 21A2 variants: CD spectra as a function of temperature. A,
wild-type P450 21A2; B, G292C; C, G292S; D, V282L; E, R409C; F, P31L; G, W303R; H, L108R. The
spectra are labeled with the temperatures. The percentage of α-helicity at 25 °C was calculated as
described (19) as modified by Chen and Yang (20): A, 20%; B, 11%; C, 19%; D, 8%; E, 20%; F, 24%; G,
1
8%; H, 14%.
2
4

Functional basis of P450 21A2 deficiencies
A
B
Wild-type
4
2.2 ± 0.6 °C
4
0.5 ± 1.0 °C
P31L
C
44.0 ± 0.6 °C
G292C
D
E
40.3 ± 0.4 °C
G292S
36.7 ± 0.4 °C
V282L
F
41.2 ± 0.9 °C
T296N
Wavelength, nm
T, °C
2
5

Functional basis of P450 21A2 deficiencies
2
+
FIGURE 6. Thermal stability of P450 21A2 variants: P450 Fe -CO spectra in bacterial cells as a
function of temperature. A, wild-type P450 21A2; B, P31L; C, G292C; D, G292S; E, V282L; F, T296N.
In each case, the spectra are shown, beginning with the red spectrum at 23 °C and with increasing
temperature as indicated by the points. The new temperature was held for 10 min after each increase,
before scanning. The plots were fit to a sigmoidal model in Prism (GraphPad) with the maximum and
minimum amount of P450 (normalized to 1.0 & 0). The estimated “T50” values (1/2 loss of P450,
corresponding to EC50 in the software, Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X))) were
calculated and are shown on the plots.
2
6

Functional basis of P450 21A2 deficiencies
FIGURE 7.
Environments of selected variants in the structure of the P450 21A2 17α-OH
progesterone complex. A, I172N (SV); B, V282L (NC); C, G292C (SW); D, G292S (SW); E, R357W
SW); F, R409C (SW). Mutated residues are highlighted with carbon atoms colored in light blue (wild-
(
type) and green (mutant) and substrate carbon atoms are colored in cyan. Amino acids in the vicinity of
mutated residues are labeled and contacts with distances of <3.3 Å are indicated by thin solid lines.
2
7

Functional Analysis of Human Cytochrome P450 21A2 Variants Involved in
Congenital Adrenal Hyperplasia
Chunxue Wang, Pradeep S. Pallan, Wei Zhang, Li Lei, Francis K. Yoshimoto, Michael R.
Waterman, Martin Egli and F. Peter Guengerich
J. Biol. Chem. published online May 24, 2017
Access the most updated version of this article at doi: 10.1074/jbc.M117.792465
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