Purification and functional characterization of human
11b hydroxylase expressed in Escherichia coli
Andy Zo
¨
llner
1
, Norio Kagawa
1,2
, Michael R. Waterman
2
, Yasuki Nonaka
3
, Koji Takio
4
,
Yoshitsugu Shiro
4
, Frank Hannemann
1
and Rita Bernhardt
1
1 Department of Biochemistry, Saarland University, Saarbru
¨
cken, Germany
2 Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, TN, USA
3 College of Nutrition, Koshien University, Takarazuka, Hyogo, Japan
4 Biometal Science Laboratory, Riken Spring-8 Center, Harima Institute, Hyogo, Japan
The final steps in the synthesis of the major human
glucocorticoid, cortisol, and the most important miner-
alocorticoid in humans, aldosterone [1], are catalyzed
by 95% identical mitochondrial cytochrome P450 iso-

reduction equivalents needed for this reaction are provided via a short elec-
tron transfer chain consisting of a [2Fe-2S] ferredoxin and a FAD-contain-
ing reductase. On the biochemical and biophysical level, little is known
about hCYP11B1 because it is very unstable for analyses performed
in vitro. This instability is also the reason why it has not been possible to
stably express it so far in Escherichia coli and subsequently purify it. In the
present study, we report on the successful and reproducible purification of
recombinant hCYP11B1 coexpressed with molecular chaperones GroES ⁄
GroEL in E. coli. The protein was highly purified to apparent homogene-
ity, as observed by SDS ⁄ PAGE. Upon mass spectrometry, the mass-to-
charge ratio (m ⁄ z) of the protein was estimated to be 55 761, which is
consistent with the value 55 760.76 calculated for the form lacking the
translational initiator Met. The functionality of hCYP11B1 was analyzed
using different methods (substrate conversion assays, stopped-flow, Bia-
core). The results clearly demonstrate that the enzyme is capable of
hydroxylating its substrates at position 11-beta. Moreover, the determined
NADPH coupling percentage for the hCYP11B1 catalyzed reactions using
either 11-deoxycortisol or 11-deoxycorticosterone as substrates was approx-
imately 75% in both cases. Biacore and stopped-flow measurements indi-
cate that hCYP11B1 possesses more than one binding site for its redox
partner adrenodoxin, possibly resulting in the formation of more than one
productive complexes. In addition, we performed CD measurements to
obtain information about the structure of hCYP11B1.
Abbreviations
ACTH, adrenocorticotrophic hormone; AdR, adrenodoxin reductase; Adx, adrenodoxin; bCYP11B1, bovine 11b hydroxylase; hCYP11B1,
human 11b hydroxylase.
FEBS Journal 275 (2008) 799–810 ª 2008 The Authors Journal compilation ª 2008 FEBS 799
and is regulated by angiotensin II and potassium, with
ACTH having mostly a short-term effect on expression
[3,4]. Interestingly, in bovine CYP11B1 (bCYP11B1),

short electron transfer chain consisting of a [2Fe-2S]
ferredoxin, adrenodoxin (Adx) and a NADPH-depen-
dent, FAD containing reductase, adrenodoxin reduc-
tase (AdR; EC 1.18.1.2) [8,9]. This electron transfer
chain is also responsible for providing electrons for the
conversion of cholesterol to pregnenolone, the precur-
sor molecule of all steroid hormones, which is pro-
duced in a reaction catalyzed by CYP11A1 [10,11].
So far, little is known about the interaction between
hCYP11B1 and its redox partner Adx. This is mainly
due not only to the scarce availability of human adre-
nals, but also to the instability of this protein, which
has hindered its expression in Escherichia coli and its
subsequent purification. Therefore, most of the studies
performed to date have been carried out using bovine
CYP11B1 in a detergent solubilized system or in lipo-
somes [12,13]. However, the instability of the solubi-
lized enzyme, mainly due to its hydrophobic nature,
has hindered any detailed investigation [13]. Moreover,
purification of the homologous bovine protein from
adrenal glands is known to be difficult, time consum-
ing and renders only small quantities of the purified
protein (4–8 mg from 1.25 g of mitochondrial pellets
[14]). In the present study, we describe the successful
expression of human CYP11B1 in E. coli as well as its
subsequent purification in significant quantities. Addi-
tionally, we were able to functionally characterize this
enzyme by using bovine Adx and AdR as electron
donors. Taking this into account, this study opens new
perspectives for the investigation of the structure and

)1
, as estimated from the
reduced CO-difference spectrum and protein assay (the
theoretical value 17.8 nmolÆmg
)1
). The purified protein
was apparently homogeneous upon SDS ⁄ PAGE
(Fig. 1) and showed a single major peak on HPLC
analysis using a POROS column (Fig. 2A). The peak
was collected and subjected to MALDI-TOF analysis.
Signals of singly (m ⁄ z = 55761) and doubly
(m ⁄ z = 27898) charged apoprotein were observed
(Fig. 2B). The m ⁄ z value is in good agreement with the
calculated molecular mass of 55760.76 for the transla-
tional initiator Met-deleted hCYP11B1.
As shown in Fig. 3, the UV ⁄ visible spectrum of
purified recombinant CYP11B1 revealed a pronounced
Soret peak at 392 nm in the absence of substrates
(Fig. 3, spectrum 1), indicating that the protein is in its
Functional characterization of hCYP11B1 A. Zo
¨
llner et al.
800 FEBS Journal 275 (2008) 799–810 ª 2008 The Authors Journal compilation ª 2008 FEBS
high spin state. The spectrum was not changed by the
addition of 17,21-hydroxyprogesterone (spectrum 2),
although the P450 was reduced by sodium dithionite
(spectrum 3) and formed the reduced CO complex
(spectrum 4) that produced a typical P450 peak of
reduced CO-difference spectrum at 448 nm (Fig. 3,
lower panel). The finding that the recombinant protein

of substrate, the signal of the positive CD band
at 290 nm and the negative band at 386 nm decreased
(Fig. 4B). A similar observation was described for sub-
strate binding of cytochrome P450RR1 from Rhodo-
coccus rhodochrous [23].
The functionality of the enzyme was demonstrated
by performing hCYP11B1-dependent substrate conver-
sion assays using 11-deoxycorticosterone and 11-de-
oxycortisol as substrate and by a subsequent HPLC
analysis of the steroid product pattern (Fig. 5). The
hCYP11B1 electron transfer chain was always reconsti-
tuted using bovine AdR and bovine Adx, which is
Purified human CYP11B1
24
µg
8
Fig. 1. SDS polyacrylamide gel electrophoresis of the purified
CYP11B1. Different amounts of the purified hCYP11B1 (2, 4, 8 lg
per lane) were separated by SDS ⁄ PAGE (10%) and visualized by
Coomassie staining.
A
0 5 10 15 20 25 20 40
27898
55761
60 80 100
Time (min) m/z (×10
3
)
B
Fig. 2. HPLC and mass spectral analysis of the purified hCYP11B1. (A) The purified hCYP11B1 was applied on RP-HPLC analysis using

sol, might be caused by the additional hydroxyl group
at position C17 of 11-deoxycortisol. This additional
OH group is likely to hinder the entrance of the
slightly more hydrophilic and bulky substrate 11-de-
oxycortisol into the active site of the enzyme.
Compared with the published values for the forma-
tion of corticosterone by the bovine enzyme,
bCYP11B1 (k
cat
= 0.1 s
)1
), the obtained values using
hCYP11B1 are approximately ten-fold higher. This
finding is quite surprising because the sequence identity
between the human enzyme and the bovine enzyme is
high (73%). However, some of the main differences
between human and bovine CYP11B1 are located in
0.15
0.10
0.05
AbsorbanceΔAbsorbance
1
2
3
4
1: Free
2: 17,21-OH-Prog
3: Reduced
4: Reduced-CO
0.00

centration of CYP11B1 was 10 l
M in 10 mM potassium phosphate
buffer, pH 7.4. Substrate was added to a concentration of 20 l
M.
Functional characterization of hCYP11B1 A. Zo
¨
llner et al.
802 FEBS Journal 275 (2008) 799–810 ª 2008 The Authors Journal compilation ª 2008 FEBS
substrate recognition sites 2 and 3 (SRS2 and SRS3),
which are mainly composed of the F and G helix of
the cytochrome [24]. This inconsistency might be the
cause for the significantly different interspecies conver-
sion rates. However, the alterations may also be
explained by the broader substrate-binding spectrum
of bCYP11B1 resulting from the combination of the
functions of CYP11B1 and CYP11B2 within one
enzyme.
Comparison of the k
cat
values obtained for the con-
version of 11-deoxycorticosterone by hCYP11B1 with
values published for corticosterone formation by rec-
ombinantly expressed and purified CYP11B1 from rats
(2.18 s
)1
) indicated that the maximal rate determined
for hCYP11B1 has not been significantly altered
(approximately 2.5-fold lower).
The k
cat

(A) Conversions of the substrate 11-deoxy-
corticosterone (DOC) to the products B (cor-
ticosterone) and 18OH-B using cortisol (F)
as internal standard. (B) shows the conver-
sion of the substrate 11-deoxycortisol (RSS)
to F using 11-deoxycorticosterone as inter-
nal standard.
A
B
k
k
m
m
Fig. 6. hCYB11B1 substrate conversion assays were performed
using the reconstituted electron transfer chain consisting of bovine
Adx and bovine AdR as well as different substrate concentrations:
11-deoxycortisol (A) and 11-deoxycorticosterone (B). Steroid separa-
tion was achieved via HPLC analysis as indicated in Experimental
procedures. V
max
values (nmol productÆmin
)1
Ænmol
)1
hCYP11B1)
were subsequently converted into k
cat
values (s
)1
).

Biacore measurements were performed to investigate
the binding behavior between bovine Adx
ox
and
hCYP11B1
ox
or bCYP11B1
ox
in more detail. Among
the binding models available in the standard software
(e.g. 1 : 1 binding or complexes with higher stoichio-
metry), the best fit was always observed with the
‘bivalent analyte’ model. This suggested that CYP11B1
possesses more than one binding site for Adx. Taking
possible steric hindrances on the chip surface into
account, only the formation of the first predominant
1 : 1 complex has been considered (Table 2), as was
the case in a previous study [25]. As seen in Table 2,
the K
D
values obtained for the predominant 1 : 1 com-
plexes for both CYP11B1 species were in the nm range.
Surprisingly, the k
on
rate of the bCYP11B1 ⁄ Adx com-
plex was two-fold slower compared with the on-rate
for the hCYP11B1 ⁄ Adx complex. On the other hand,
the off rate was five-fold faster for the hCYP11-
B1 ⁄ Adx, indicating a slightly weaker stability com-
pared to the physiological interaction.

heterogenous sample composition. Since it is known
that the Adx concentration plays a role in the regula-
tion of the activity of CYP11A1 [26], CYP11B1 [27]
and CYP11B2 [28], this finding is not surprising. The
maximal velocities extracted from the plots shown in
Fig. 7B,C for the second and third phase were 3.89 s
)1
and 0.65 s
)1
, respectively. The K
D
values obtained
from these experiments for the interaction between the
relevant redox states of Adx and hCYP11B1 were
0.78 lm for the second phase and 2.2 lm for the third
phase. Since the K
D
value obtained from the optical
Table 1. Kinetic parameters obtained for the conversion of 11-deoxycorticosterone or 11-deoxycortisol using different Adx concentrations
under substrate saturation (left). Kinetic parameters obtained using different substrate concentrations are also shown (right).
Substrate
Adx-dependent Substrate-dependent
K
m
(lM) k
cat
(s
)1
) K
m

Functional characterization of hCYP11B1 A. Zo
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804 FEBS Journal 275 (2008) 799–810 ª 2008 The Authors Journal compilation ª 2008 FEBS
biosensor measurements and the value obtained for the
productive complex leading to the second reaction
phase are in the same range, it can be assumed that
this complex is the predominant complex seen with the
Biacore device. Moreover, analysis of the amplitude
change of these phases extracted from the stopped-flow
experiments indicates that the second complex is fur-
ther stabilized in the presence of increasing amounts of
Adx, whereas the complex leading to the third reaction
phase is favored in the presence of small amounts of
Adx (Fig. 7). These findings suggest different thermo-
dynamic attributes for the productive complexes.
Nevertheless, it cannot be ruled out that the differ-
ent reaction phases observed in these experiments are
caused by complex rearrangements or conformational
gating that might be necessary before an efficient elec-
tron transfer can take place. More investigations
including Adx and CYP11B1 mutants will be necessary
to investigate this question in more detail. However,
considering the current data, it is very likely that a
productive interaction (complex formation) between
Adx and CYP11B1 can take place through more than
one productive complex, as previously postulated for
the interaction between bovine CYP11B1 and Adx
[29].
Moreover, comparison of the k

can enable such high turnover rates. Additionally, it
appears that the second and the third phase seen in
the stopped-flow measurements are negligible during
the hydroxylation reaction, although possessing a
higher amplitude change in the stopped-flow measure-
ments compared to phase 1. Otherwise, the k
cat
values
from the substrate conversion assays were likely to be
in the range of the k
obs,max
values obtained for these
phases. However, more investigations are necessary to
clarify this assumption. In this context, it is possible
that the predominant, but slower phases observed in
the stopped-flow experiments play a role in the regula-
tion of the activity of CYP11B1, especially since the
absolute amplitude change of these phases when using
higher Adx concentrations increases and an involve-
ment of the Adx concentration in the regulation of
CYP11B1 has been demonstrated previously [27]. Nev-
ertheless, the physiological relevance of these different
phases and the postulated different complexes remains
unclear and should be subject of further studies.
In summary, the present study reports on the suc-
cessful purification of functional hCYP11B1 expressed
in E. coli. This will open new possibilities for analyzing
this very important cytochrome P450 in vitro, including
the detailed investigation of the interaction of
hCYP11B1 with its redox partner, Adx. As indicated

C-terminus to facilitate the purification. The CYP11B1
expression plasmid was introduced into E. coli strain
BL21(DE3)pLys along with a GroES ⁄ GroEL expression
vector pGro12 [31].
The human CYP11B1 was expressed and extracted from
E. coli in a similar manner to the methods previously
described for the expression of CYP19 and CYP21 [18].
The extracts (100 mL) from 1 L culture were applied on a
Ni-NTA agarose (10 mL bed volume) column equilibrated
with buffer A (50 mm potassium phosphate, pH 7.4,
500 mm sodium acetate, 20% glycerol, 0.1 mm EDTA,
0.1 mm dithiothreitol, 1% sodium cholate, 1% Tween 20,
0.1 mm phenylmethanesulfonyl fluoride), washed with
75 mL of buffer A plus 40 mm imidazole, and with
20 mL of buffer B (50 mm potassium phosphate, pH 7.4,
20% glycerol, 0.1 EDTA, 0.1 mm dithiothreitol,
40 mm imidazole, 1% sodium cholate, 1% Tween 20,
0.1 mm ATP and 0.1 mm phenylmethanesulfonyl fluoride).
Proteins were eluted with buffer C (200 mm imidazole
acetate, pH 7.4, 20% glycerol, 0.1 mm EDTA, 0.1 mm di-
thiothreitol, 1% sodium cholate, 1% Tween 20). The red-
colored fractions were combined and diluted with five
volumes of buffer D (20% glycerol, 0.1 mm EDTA,
0.1 mm dithiothreitol, 1% sodium cholate, pH 7.4) and
applied on a DEAE-Sepharose (30 · 50 mm) equilibrated
with buffer E (20 mm potassium phosphate, pH 7.4,
20% glycerol, 0.1 mm EDTA, 0.1 m m dithiothreitol,
10 mm imidazole, 1% sodium cholate, 0.1% Tween 20).
The column was washed with 40 mL of buffer E. Pass-
through fractions were then applied on a SP-Sepharose

Applied Biosystems, Foster City, CA, USA) using a liquid
chromatograph (Agilent model 1100; Agilent Technologies,
Palo Alto, CA, USA) with a 16 min linear gradient of
8–72% CH3CN in 0.1% trifluoroacetic acid at a flow rate
of 0.1 mLÆmin
)1
. Column effluent was monitored by absor-
bance at 215 nm, 254 nm, 275 nm, 290 nm and 400 nm.
The peak eluted at 13.5 min contained the heme extracted
from h11B1 and that at 15.5 min contained the apoprotein.
The apoprotein peak was collected and subjected to
MALDI-TOF MS to verify the integrity of the protein
moiety on Voyager DE-Pro (Applied Biosystems) with sina-
pic acid as matrix.
UV/visible and CD spectroscopy
UV ⁄ visible spectra of CYP11B1 were recorded at room
temperature on a Shimadzu double-beam spectrophoto-
meter (UV2100PC; Shimadzu, Kyoto, Japan). The con-
centration of the 11b-hydroxylase was estimated by carbon
monoxide difference spectra assuming e
450–490
=
91 mm
)1
Æcm
)1
according to [35]. Adx and AdR concentra-
tions were determined using the molar extinction coefficient
e
415

ity depending on the Adx concentration were performed as
previously described for CYP11A1 reconstitution assays
[38] with slight modifications. All experiments were per-
formed using bovine Adx, which is capable of interacting
with hCYP11B1. Bovine and human Adx exhibit a
90% primary structure identity. Briefly, the reaction mix-
ture (0.5 mL) consisted of CYP11B1 (0.4 lm), AdR
(0.5 lm), Adx (2–20 lm), 11-deoxycortisol or 11-deoxycorti-
costerone (400 lm), MgCl
2
(1 mm)in50mm Hepes buffer
(pH 7.3, 0.05% (v ⁄ v) Tween 20). In addition to this, a
NADPH regenerating system consisting of MgCl
2
(1 lm),
glucose 6-phosphate (5 lm) and glucose 6-phosphate dehy-
drogenase (1 U) was applied.
In another set of experiments, we varied the substrate
concentration in the range 0–700 lm for both substrates
whereas the Adx concentration was fixed at 10 lm. All other
components were as described above. All reactions were ini-
tiated by the addition of 100 lm NADPH and were carried
out for 10 min at 37 °C. After stopping the reaction by add-
ing chloroform, either cortisol (for 11-deoxycorticosterone
conversion assays) or 11-deoxycorticosterone (for 11-deoxy-
cortisol conversion) was added to the corresponding reac-
tion mixture as an internal standard. After extraction of the
steroids and evaporation of the chloroform phase, the ste-
roids were resuspended in 200 lL acetonitrile and separated
on a Jasco reversed phase HPLC system of the LC900 series

hCYP11B1) were subsequently converted into
k
cat
values (s
)1
).
To correlate the NADPH consumption with the amount
of product formed (i.e. the coupling percentage), we per-
formed additional experiments. Samples generated for
this purpose contained 400 l m substrate (11-deoxycortisol
or 11-deoxycorticosterone), 0.4 lm hCYP11B1, 3 lm Adx,
0.5 lm AdR in 50 mm Hepes buffer, pH. 7.4, containing
0.05% Tween 20. The reaction was initiated by addition of
NADPH to a final concentration of 100 lm. Reaction con-
ditions were as described above. The sample volume was
500 lL. NADPH consumption was determined spectro-
scopically by recording the absorption changes of the
A. Zo
¨
llner et al. Functional characterization of hCYP11B1
FEBS Journal 275 (2008) 799–810 ª 2008 The Authors Journal compilation ª 2008 FEBS 807
reaction mixture at 340 nm, corresponding to the absorp-
tion maximum of NADPH, at the start of the reaction
(t = 0) and after 10 min. To subtract protein absorption at
this wavelength, we used a reference reaction sample with-
out NADPH. NADPH consumption (i.e. the amount
of NADPH consumed during the reaction) was sub-
sequently determined by using the Lambert–Beer law
(e
340

after injection of solutions with varying concentrations in
the range 10–500 nm. Each concentration was injected at
least three times. To visualize unspecific background inter-
actions between the dextran matrix and CYP11B1, a refer-
ence cell was created. To remove the bound CYP11B1,
10 lLof a 2mm NaOH solution was injected. K
D
values
were determined using the software biaeval, version 3.1.
Averaged binding curves for the interaction between Adx
and varying CYP11B1 concentrations were fitted simulta-
neously using different binding models available in the eval-
uation software (e.g. 1 : 1 Languimir-binding or a bivalent
binding model as at least two possible interaction sites for
Adx exist on CYP11B1). K
D
values were determined from
the fit with the lowest standard deviation.
Kinetics by rapid mixing
Stopped flow measurements were carried out on a SFM 300
stopped-flow spectrophotometer equipped with a FC 100 ⁄ 10
cuvette and a MPS 60 data-processing unit (Biologic SAS,
Claix, France) at 15 °C. Anaerobic conditions were achieved
by incubation of the stopped-flow device for 20 min with
argon-bubbled buffer containing 5 mm dithionite followed
by repeated flushing with excessively Ar-bubbled reaction
buffer to remove oxygen and remaining dithionite from the
system. All samples were prepared in a glove box in an oxy-
gen-free atmosphere. The reaction buffer applied for all mea-
surements was a 50 mm Hepes buffer (pH 7.4) containing

MRW. The authors would like to thank K. Neumann,
A. Eiden-Plach and W. Reinle for their excellent tech-
nical support.
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