Baboon cytochrome P450 17a-hydroxylase/17,20-lyase (CYP17)
Characterization of the adrenal microsomal and cloned enzymes
Amanda C. Swart
1
, Norbert W. Kolar
1
, Nic Lombard
1
, J. Ian Mason
2
and Pieter Swart
1
1
Department of Biochemistry, University of Stellenbosch, South Africa;
2
Department of Reproductive & Developmental Sciences,
University of Edinburgh Medical School, Scotland, UK
Human cytochrome P450 17a-hydroxylase (CYP17) cata-
lyses not only the 17a-hydroxlation of pregnenolone and
progesterone and the C17,20-side chain cleavage (lyase) of
17a-hydroxypregnenolone, necessary for the biosynthesis of
C
21
-glucocorticoids and C
19
-androgens, but also catalyses
the 16a-hydroxylation of progesterone. In efforts to under-
stand the complex enzymology of CYP17, structure/func-
tion relationships have been reported previously after
expressing recombinant DNAs, encoding CYP17 from
various species, in nonsteroidogenic mammalian or yeast

lyase activity for 17a-hydroxyprogesterone. Sequence ana-
lyses showed that there are 28 different amino acid residues
between human and baboon CYP17, primarily in helices F
and G and the F-G loop.
Keywords: CYP17; baboon; cytochrome P450; 17a-
hydroxylase; 17, 20-lyase.
The steroidogenic cytochromes P450 are a unique group of
enzymes responsible for the synthesis of hormones vital for
reproduction, stress management and the control of water
and mineral balance in mammals. These enzymes catalyse
the biosynthesis of mineralocorticosteroids, glucocortico-
steroids and androgens and although the steroidogenic
cytochromes P450 share acommon reaction mechanism with
their counterparts in organs like the liver and the lung, they
are substantially more substrate and organ specific. Within
the ambit of the steroidogenic cytochromes P450, cyto-
chrome P450 17a-hydroxylase (CYP17) catalyses at least two
distinctly different reactions, the 17a-hydroxylase and the
17,20-lyase reactions, of C
21
-steroids, placing this enzyme at
a key branch point in the biosynthesis of aldosterone, cortisol
and androgens. The 17a-hydroxylation of the D
5
-andD
4
-
steroids, pregnenolone (PREG) and progesterone (PROG),
yields 17a-hydroxypregnenolone (17-OHPREG) and
17a-hydroxyprogesterone (17-OHPROG), respectively.

from its electron-transfer partner, FAD/FMN-dependent
Correspondence to A. C. Swart, Department of Biochemistry,
University of Stellenbosch, Private Bag X1, Matieland, 7602,
South Africa. Fax: + 27 21 8085863, Tel.: + 27 21 8085862,
E-mail:
Abbreviations: PROG, progesterone; 17-OHPROG, 17a-hydroxy-
progesterone; 16-OHPROG, 16a-hydroxyprogesterone; PREG,
pregnenolone; 17-OHPREG, 17a-hydroxypregnenolone; 3b-HSD,
3b-hydroxysteroid dehydrogenase/D5-D4 isomerase; A4, 4-andros-
tene-3,17-dione; DEPC, diethylpyrocarbonate; DHEA, dehydro-
epiandrosterone; DHEA-S, dehydroepiandrosterone-sulphate; DOC,
deoxycorticosterone; CYP17, cytochrome P450 17a-hydroxylase;
CYP21, cytochrome P450 21-hydroxylase; CYP11A, cytochrome
P450 side chain cleavage; CYP11B1, cytochrome P450 11b-hydroxy-
lase; ACTH, adrenocorticotrophic hormone.
Enzymes: cytochrome P450 17a-hydroxylase (CYP17) EC 1.14.99.9.
(Received 6 July 2002, revised 5 September 2002,
accepted 18 September 2002)
Eur. J. Biochem. 269, 5608–5616 (2002) Ó FEBS 2002 doi:10.1046/j.1432-1033.2002.03268.x
NADPH-cytochrome P450 reductase. The production of
C
19
androgen precursors from the 17a-hydroxy intermedi-
ates involves another two rounds of mono oxygenation. The
availability of reducing equivalents enhances the lyase
activity of CYP17 and it would appear that high expression
levels of cytochrome b
5
also increases the biosynthesis of
androgens in some species [3,4].

-andD
4
-steroids is quite similar
across species, but notable differences exist in the ability of
the enzyme to cleave 17-OHPREG and 17-OHPROG.
Human and bovine CYP17 catalyse the hydroxylation
of both PREG and PROG and the conversion of
17-OHPREG to DHEA but lyase activity for the 17-OH-
PROG intermediate is negligible [11,12]. The hydroxylase
and lyase activity of guinea pig CYP17 favours the
D
4
-steroid pathway, the enzyme being incapable of meta-
bolizing 17-OHPREG to DHEA [13]. Rat, porcine and
hamster CYP17 catalyse the D
4
and D
5
hydroxylase and
lyase reactions, yielding both DHEA and A4 [14–16].
The alignment of mammalian cytochromes P450 with
bacterial cytochromes P450 has allowed the prediction of
domains involved in substrate binding and redox partner
interaction [10,17]. Although site-directed mutageneses and
naturally occurring CYP17 mutants have pinpointed specific
amino acid residues playing an essential role in structure/
function relationship of CYP17, interspecies homology
alignments of CYP17 have been less effective in structure/
function analysis [18]. The characterization of baboon
cytochrome CYP17 would allow comparative studies of

For all experiments material was collected from 20 groups
consisting of between two and four baboons over a period
of eight years. All the experiments to be described were
carried out on at least three different groups of adrenals.
Reagents
[
3
H]PROG, [
3
H]17-OHPROG, DHEA and A4 were pur-
chased from Amersham Life Science (Amersham, Bucks,
UK) and [
3
H]PREG from Dupont New England Nuclear
(Boston, MA, USA). Antibiotics, NADPH and diethyl-
aminoethyl-dextran were purchased from Sigma Chemical
Co. (St Louis, MO, USA). Bacterial culture media were
purchased from Difco Laboratories (Detroit, MI, USA)
and tissue culture media from Gibco-BRL (Gaithersburg,
MD, USA). Plasmid vectors, restriction enzymes, T4
Ligase, Taq DNA polymerase were purchased from
Promega Bioteck (Madison, WI, USA) and ribonucleotide
triphosphates from Boehringer Mannheim Biochemicals
(Mannheim, Germany). All other chemicals were of reagent
grade purchased from scientific supply houses.
Determination of the cytochromes P450 and
b
5
contents of baboon adrenal microsomes
Microsomes were prepared from baboon adrenal cortex

(10 l
M
)inatotalvolumeof0.5mLfor5minat37 °C. The
reaction was initiated by the addition of NADPH
(11 nmol). An aliquot (50 lL) of the reaction mixture was
removed prior immediately to the addition of NADPH and
subsequently at 2-min intervals. The same protocol was
followed to assay 17-OHPROG metabolism in baboon
adrenal microsomes. Steroids were extracted with dichloro-
methane (10 volumes), the dichloromethane phase was
evaporated under N
2
and the dried residue redissolved in
methanol prior to HPLC analysis.
Separation and quantification of steroids
Chromatography was performed on a Waters (Milford,
MA, USA) high performance liquid chromatograph cou-
pled to a WISP
TM
automatic injector (Waters) and a Flo-
One liquid scintillation spectrophotometer (Radiomatic,
Tampa, FL). PROG metabolites were separated on a
NovapakÒ C
18
column at a flow rate of 1 mLÆmin
)1
.The
mobile phase consisted of solvent A (water/methanol 45/55)
and solvent B (100% methanol). The column was eluted for
15 min with solvent A, followed by a linear gradient from

CYP17, 5¢-tagtctcgagtactgtctatcttgcctgctga-3¢ (sense), and
5¢-tatacccgggaagcttttaggtgctaccctcagcctg-3¢ (antisense) were
used. The RT-PCR product was gel purified, digested with
XhoI and cloned into a mammalian expression vector, pCI-
neo, previously digested with XhoIandSmaI. Nucleotide
sequences of both strands, purified RT-PCR product and
cloned cDNA, were determined using the Bigdye
TM
Version
2 diterminator sequencing kit (model 373 A ABI, Applied
Biosystems, Foster City, CA).
Assay of CYP17 enzyme activity in
HEK-293 cells
HEK-293 cells, grown in Dulbecco’s modified Eagle’s
medium (DMEM), containing 0.9 gÆL
)1
glucose, 0.12%
NaHCO
3
, 10% fetal bovine serum, 1% penicillin-strepto-
mycin were transfected with the pCI-neo/baboon CYP17
construct, 5 lgÆmL
)1
, using diethylaminoethyl-dextran,
0.25 mgÆmL
)1
, with the later addition of 100 l
M
chloro-
quine [24]. The same protocol was followed to determine the

NADH to the reference cuvette (Fig. 1B). The NADH
reduced vs. oxidized difference spectrum of baboon adrenal
microsomes is given in Fig. 1C. The spectrum with a
maximum at 424 nm and a minimum at 409 nm is charac-
teristic of cytochrome b
5
[20]. The concentration of baboon
adrenal cytochrome P450 was 0.55 nmolÆmg
)1
microsomal
protein and the concentration of cytochrome b
5
was 0.17 nmolÆmg
)1
protein.
Fig. 1. Carbon monoxide dithionite reduced
vs. oxidized difference spectrum of baboon
adrenal microsomal cytochrome P450. (A)
Before the addition and (B) after the addition
of NADH to the reference cuvette. The
reduction in the peak at 425 nm after addi-
tion of NADH is indicative of the presence of
cytochrome b
5
. (C) NADH reduced vs.
oxidized difference spectrum of ovine adrenal
microsomes. The maximum at 424 nm and a
minimum at 404 nm is indicative of cyto-
chrome b
5

metabolism (10 l
M
) by baboon adrenal
microsomes at 4 min (A) and at 15 min (B).
Peaks on the chromatogram are: 1, PROG
(25.75 min); 2, 17-OHPROG (14 min); 3,
DOC (11 min); and 4, deoxycortisol
(6.5 min).
Fig. 4. Metabolism of 17-OHPROG (10 l
M
) by baboon adrenal
microsomes (0.5 l
M
P450).
Ó FEBS 2002 Characterization of baboon CYP17 (Eur. J. Biochem. 269) 5611
CYP17 (GenBank accession no. AY 034635), yielded a
single 1524 bp product which was cloned and sequenced.
The nucleotide sequence (GenBank accession no. AF
297650) showed 96% homology with human CYP17
cDNA and encodes for a predicted 508 amino acid
protein. The 28 amino acid differences between baboon
and human CYP17 are predominantly conservative with
some differences resulting in a change in side chain size and
polarity. Exon 3 and 4 show the least homology between the
baboon and human sequence. Significant changes include
three positively charged residues K196, H199 and R234
which correspond to polar residues in the human sequence
and the two larger aromatic residues, F218 and F247, in the
baboon sequence which correspond to S and L in the
human sequence. In exon 7 there is another positively

and V values for PREG utilization by
baboon CYP17 were 0.9 l
M
and 0.45 nmolÆh
)1
Æmg
)1
pro-
tein, respectively (Fig. 8). These values did not differ
significantly from the values obtained for human CYP17
under the same circumstances (Table 1).
The metabolism of PROG by baboon CYP17 expressed
in HEK-293 cells yielded only 17-OHPROG (Fig. 9).
PROG metabolism by human CYP17 expressed under the
same conditions, yielded 17-OHPROG, 16a-hydroxypro-
gesterone but no A4 (Fig. 10). The ratio of 17-OHPROG to
16a-hydroxyprogesterone was approximately 4 : 1 as pre-
viously reported for expression in COS 1 cells [1]. The K
m
for PROG utilization by baboon CYP17 expressed in HEK-
293 cells, was 6.5 l
M
and the maximum velocity (V value)
was 3.9 nmolÆh
)1
Æmg
)1
protein (Fig. 11). As reflected in the
V value of the two enzymes, HEK-293 cells expressing
baboon CYP17 utilized PROG at a higher rate than

and amino acid residues crucial to the catalytic activity of
the enzyme. The interspecies differences, however, make it
difficult to extrapolate nonprimate and rodent data on
steroid metabolism in primates and subsequently complicate
deductions pertaining to structure/function relationships.
Our report describes the molecular and enzymatic charac-
terization of CYP17 in the Cape baboon, a species closely
related to humans. Baboon CYP17 encodes a deduced
protein of 508 amino acid residues exhibiting, in primary
structure, 96% sequence similarity to that of human
CYP17. Baboon CYP17 exhibited distinct differences and
Table 1. Summary of kinetics of PROG and PREG metabolism by baboon CYP17 expressed in HEK-293 cells. For each substrate concentration,
initial reaction rates of PROG and PREG utilization were determined at various substrate concentrations by linear regression. At least five time
points were used for each rate determination and in the cases where a slight lag phase was observed, only the linear part of the curve was used. The
R-squared value for all initial rate regression analyses was always higher than 0.98. K
m
values are the mean ± SEM of three experiments.
Progesterone Pregnenolone
Species K
m
(l
M
) V (nmolÆh
)1
Æmg protein
)1
) K
m
(l
M

influenced, not only by the cellular lipid environment, but
also by the presence of electron transport proteins,
cytochrome P450 reductase and cytochrome b
5
.Inaddi-
tion 3b-HSD competes with CYP17 for the same
substrates, PREG and 17-OHPREG, while CYP21 com-
petes with CYP17 for PROG and 17-OHPROG. Our
experiments with baboon adrenal microsomes enabled the
investigation of baboon CYP17 activity in the physiolo-
gical environment of the endoplasmic reticulum. In the
baboon adrenal microsomal preparations, the PROG
17-hydroxylase activity was considerably higher than the
PROG CYP21 activity as indicated by the 3 : 1 ratio of
the metabolites, deoxycortisol to DOC after all the PROG
had been utilized. In contrast, CYP17 and CYP21 of
human fetal adrenal microsomes exhibited comparable
hydroxylase activities for PROG [1]. Furthermore, human
CYP17 catalysed the formation of 16-OHPROG, a
metabolite not detected during the metabolism of PROG
by baboon CYP17. A4 was also not detected as a product
of PROG metabolism indicating that baboon CYP17, like
human CYP17, has little, if any, lyase activity towards
17-OHPROG. Cytochrome b
5
, a modulating agent of
CYP17 activity, was present in the baboon adrenal
Fig. 9. Time course of PROG (1 l
M
) metabolism by baboon CYP17

two species. Neither enzyme had lyase activity towards
PROG or 17-OHPROG but human CYP17 could convert
PROG to 16-OHPROG while the baboon enzyme could
not. To further investigate these findings the cDNA
encoding baboon CYP17 was subsequently expressed in
HEK-293 cells. PREG metabolism by baboon CYP17
expressed in HEK-293 cells, did not differ significantly from
human CYP17 expressed in the same system and the
apparent K
m
and V values for the two enzymes with PREG
as substrate, did not show a notable difference (Table 1).
The conversion of PREG to DHEA, however, appears to
differ with respect to the interaction of the enzyme with the
17-hydroxylated intermediate. Baboon CYP17 initially
converted most of the PREG to 17-OHPREG and during
the entire experiment, the ratio of DHEA/17-OHPREG
was lower than for the human enzyme while PREG was still
available as substrate (Fig. 7). In comparison, human
CYP17 metabolizes 17-OHPREG at a significantly faster
rate in the presence of PREG and the DHEA/17-OHPREG
is higher for the human CYP17 throughout the 12 h
incubation period (Fig. 7). These results indicate that
PREG could potentially have a greater influence on the
lyase activity of baboon CYP17 than on the human enzyme
and that human CYP17 possibly converts a greater
percentage of bound 17-OHPREG to DHEA. It may well
be that the 17-hydroxylated intermediate is less tightly
bound to the baboon enzyme and a greater percentage of
the 17-OHPREG will therefore leave the active site. Clearly

ling and structural alignments with bacterial cytochromes
P450 has identified domains in the primary sequence of
human CYP17 which are involved in the catalytic activity of
the enzyme, i.e. substrate docking and binding, the active
site including the heme-binding domain and redox partner
binding domain [10]. Baboon and human CYP17 are
excellent candidates for identifying regions in the primary
sequence that contribute to substrate specificity, affinity and
binding. The two species share 96% sequence similarity in
primary sequence yet baboon CYP17 seemingly has a
considerably higher apparent K
m
for PROG and no 16a-
hydroxylase activity.
Sequence alignments of CYP17 based on the structures of
bacterial cytochromes P450 (according to the alignment of
Graham-Lorence [10]) show that the most significant
differences in the primary sequences of human and baboon
CYP17 lie in the predicted substrate access and binding
regions which include helices F and G and the F-G loop.
The differences between the two species in the F and G helix
and F-G loop could alter substrate affinity by tighter
binding and the larger hydrophobic residues could change
the shape of the active pocket. It was shown by Beaudoin
et al. that guinea pig CYP17 preferentially converts PROG
to A4 and by changing a single residue arginine (R) to
asparagine (N) at position 200 in the F-helix, the substrate
specificity could be changed [28]. Introducing this specific
mutation increased the activity towards PREG. It is in this
region (residues 196–200) that there are distinct differences

J.N. & Akhtar, M. (1997) Interaction of human CYP17 (P450
17a
,
17a-hydroxylase-17,20-lyase) with cytochrome b
5
: importance of
the orientation of the hydrophobic domain of cytochrome
b
5
. Biochem. J. 321, 857–863.
Ó FEBS 2002 Characterization of baboon CYP17 (Eur. J. Biochem. 269) 5615
5. Nakajin, S., Shively, J.E., Yuan, P. & Hall, P.F. (1981) Micro-
somal cytochrome P450 from neonatal pig testis: two enzymatic
activities (17a-hydroxylase and C17,20-lyase) associated with one
protein. Biochemistry. 20, 4037–4042.
6. Biason-Lauber, A., Leiberman, E. & Zachmann, M. (1997) A
single amino acid substitution in the putative redox partner-
binding site of P450c17 as cause of isolated 17,20-lyase deficiency.
J. Clin. Endocrinol. Metab. 82, 3807–3812.
7. Fardella, C.E., Hum, D.W., Homoki, J. & Miller, W.L. (1994)
Point mutation of Arg440 to His in cytochrome P450c17 causes
severe 17a-hydroxylase deficiency. J. Clin. Endocrinol. Metab. 79,
160–164.
8. Suzuki, Y., Nagashima, T., Nomura, Y., Onigata, K., Nagashima,
K. & Morikawa, A. (1998) A new compound heterozygous
mutation (W17X, 436+5GsT) in the cytochrome P450c17 gene
causes 17a-hydroxylase/17,20-lyase deficiency. J. Clin. Endocrinol.
Metab. 83, 199–202.
9. Yanase, T., Simpson, E.R. & Waterman, M.R. (1991) 17a-
hydroxylase/lyase deficiency from clinical investigation to

hamster adrenal cytochrome P450c17 cDNA. Ann. N. Y. Acad.
Sci. 774, 294–296.
17. Negishi, M., Uno, T., Darden, T.A., Sueyoshi, T. & Pedersen,
L.G. (1996) Structural flexibility and functional versatility of
mammalian P450 enzymes. FASEB J. 10, 683–689.
18. Auchus, R.J., Worthy, K., Geller, D.H. & Miller, W.L. (2000)
Probing structural and functional domains of human P450c17.
Endocr Res. 26, 695–703.
19. Mason, J.I., Carr, B.R. & Murray, B.A. (1987) Imidazole anti-
mycotics: selective inhibitors of steroid aromatization and pro-
gesterone hydroxylation. Steroids. 50, 179–189.
20. Omura, R. & Sato, T. (1964) The carbon monoxide-binding pig-
ment of liver microsomes. I. Evidence for its hemoprotein nature.
J. Biol. Chem. 239, 2370–2378.
21. Purvis, J.L., Canick, J.A., Mason, J.I., Estabrook, R.W. &
McCarthy, J.L. (1973) Ann. NY Acad. Sci. 212, 319–343.
22. Swart, P., Engelbrecht, Y., Bellstedt, D.U., deVilliers, C.A. &
Dreesbeimdieke, C. (1995) The effect of cytochrome b
5
on pro-
gesterone metabolism in the ovine adrenal. Endocrinol. Res. 21,
297–306.
23. Sambrook, J., Fritsch, E.F. & Maniatis, T. (1980) Molecular
Cloning: a Laboratory Manual. Cold Spring Harbour Laboratory
Press, Cold Spring Harbour, New York.
24. Bradshaw, K.D., Waterman, M.R., Couch, R.T., Simpson, E.R.
& Zuber, M.X. (1987) Characterization of complementary
deoxyribonucleic acid for human adrenocortical 17a-hydroxylase:
a probe for analysis of 17a-hydroxylase deficiency. Mol. Endocr-
inol. 1, 348–354.