The characterization of baboon cytochrome CYP17 would allow comparative studies of human CYP17 with a species much more closely related than investigated thus far, and may contribute to
Trang 1Baboon cytochrome P450 17a-hydroxylase/17,20-lyase (CYP17)
Characterization of the adrenal microsomal and cloned enzymes
Amanda C Swart1, Norbert W Kolar1, Nic Lombard1, J Ian Mason2and Pieter Swart1
1
Department of Biochemistry, University of Stellenbosch, South Africa;2Department 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
C21-glucocorticoids and C19-androgens, but also catalyses
the 16a-hydroxylation of progesterone In efforts to
under-stand the complex enzymology of CYP17,
structure/func-tion relastructure/func-tionships have been reported previously after
expressingrecombinant DNAs, encodingCYP17 from
various species, in nonsteroidogenic mammalian or yeast
cells A major difference between species resides in the lyase
activity towards the hydroxylated intermediates and in the
fact that the secretion of C19-steroids take place, in some
species, principally in the gonads Because human and higher
primate adrenals secrete steroids, CYP17 has been
charac-terized in the Cape baboon, a species more closely related to
humans, in an effort to gain a further understanding of the reactions catalysed by CYP17 Baboon and human CYP17 cDNA share 96% homology Baboon CYP17 has apparent
Km and V values for pregnenolone and progesterone of 0.9 lM and 0.4 nmolÆh)1Æmgprotein)1 and 6.5 lM and 3.9 nmolÆh)1Æmgprotein)1, respectively Baboon CYP17 had
a significantly higher activity for progesterone hydroxyla-tion relative to pregnenolone No 16a-hydroxylase and no 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 a common 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 C21-steroids, placingthis enzyme at
a key branch point in the biosynthesis of aldosterone, cortisol and androgens The 17a-hydroxylation of the D5- and D4 -steroids, pregnenolone (PREG) and progesterone (PROG), yields 17a-hydroxypregnenolone (17-OHPREG) and 17a-hydroxyprogesterone (17-OHPROG), respectively The 17,20-lyase reaction cleaves the C17,20 bond converting these hydroxylated intermediates to dehydroepiandroster-one (DHEA) or androstenedidehydroepiandroster-one (A4) Both activities arise from a common active site although the precise mechanism
of catalysis is not known In addition to 17-hydroxylase and lyase activities, CYP17 also exhibits species specific steroid hydroxylase activities In humans, for instance, CYP17 also has the ability to convert PROG to 16a-hydroxyprogester-one (16-OHPROG) [1] Glucocorticoid biosynthesis requires the release of the 17a-hydroxylated product, 17-OHPROG, from the active site and subsequent hydroxylation by cytochrome P450 21-hydroxylase (CYP21) It is, however, possible for the 17a-hydroxylated intermediate to remain bound to the enzyme or for the released product to rebind for the 17,20-lyase reaction, to yield C19-steroids Androgen biosynthesis is not restricted to the gonads in humans and higher primates as DHEA and A4 are synthesized by the adrenal gland Lower vertebrates such as rodents are unable
to synthesize adrenal C19-steroids as CYP17 is not expressed
in the adrenal The activity of CYP17 is not only influenced
by the environment in which the enzyme is expressed, but also by redox partner and/or accessory proteins The 17a-hydroxylation/lyase reactions are standard mixed-func-tion oxidamixed-func-tion reacmixed-func-tions dependent on the accessibility of electron transport proteins [2] The hydroxylation reaction requires molecular oxygen and the input of two electrons from its electron-transfer partner, FAD/FMN-dependent
Correspondence to A C Swart, Department of Biochemistry,
University of Stellenbosch, Private BagX1, Matieland, 7602,
South Africa Fax: + 27 21 8085863, Tel.: + 27 21 8085862,
E-mail: acswart@sun.ac.za
Abbreviations: PROG, progesterone; 17-OHPROG,
17a-hydroxy-progesterone; 16-OHPROG, 16a-hydroxy17a-hydroxy-progesterone; 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)
Trang 2NADPH-cytochrome P450 reductase The production of
C19androgen precursors from the 17a-hydroxy
intermedi-ates involves another two rounds of mono oxygenation The
availability of reducingequivalents enhances the lyase
activity of CYP17 and it would appear that high expression
levels of cytochrome b5 also increases the biosynthesis of
androgens in some species [3,4]
The dual activity of CYP17 and the differential regulation
thereof have been the subject of many studies It is apparent
that the normal expression and dual hydroxylase and lyase
activities of this key enzyme are essential for normal
metabolic and reproductive activities in all mammals,
includingman There appears to be only one form of
CYP17, encoded by a single gene, expressed in both the
adrenals and gonads [5] Mutations in the human gene result
in the loss of a functional protein eliminating, in some
clinical cases, only the 17,20-lyase activity [6] or both the
17a-hydroxylase and 17,20-lyase activities [7,8]
Cyto-chrome P450 17a-hydroxylase deficiency is characterized
by impaired cortisol production resultingin the
hypersecre-tion of ACTH and an increased biosynthesis of
deoxycor-ticosterone (DOC) and cordeoxycor-ticosterone The absence of lyase
activity in humans results in the development of abnormal
secondary sex characteristics, sexual infantilism in females
and failure of virilization in males [7,9]
As CYP17 has not been crystallized, most information
about the structure/function relationship of this
hemopro-tein has been obtained from comparative studies between
different species, analysis of the CYP17 gene of individuals
with CYP17 deficiency as well as from homology
align-ments with bacterial cytochromes P450 [10] The degree of
amino acid sequence homology between cytochromes P450
of humans and other species ranges from 48 to 71%,
resultingin the prediction of domains playinga catalytic
role rather than specific amino acids The hydroxylase
activity of CYP17 for the D5- and D4-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
D4-steroid pathway, the enzyme beingincapable of
meta-bolizing17-OHPREG to DHEA [13] Rat, porcine and
hamster CYP17 catalyse the D4 and D5 hydroxylase and
lyase reactions, yieldingboth DHEA and A4 [14–16]
The alignment of mammalian cytochromes P450 with
bacterial cytochromes P450 has allowed the prediction of
domains involved in substrate bindingand redox partner
interaction [10,17] Although site-directed mutageneses and
naturally occurringCYP17 mutants have pinpointed specific
amino acid residues playingan 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
human CYP17 with a species much more closely related than
investigated thus far, and may contribute to a further
understandingof the hydroxylase and lyase activities of the
enzyme in relation to substrate bindingand orientation
PROG and 17-OHPROG metabolism was investigated in
baboon adrenal microsomes to determine the catalytic
properties of baboon CYP17 in the presence of the compet-ingCYP21 enzyme and of membrane components including NADPH-cytochrome P450 reductase, cytochrome b5 and phospholipids The gene encoding baboon CYP17 was isolated from baboon adrenal mRNA The recombinant DNA was expressed in nonsteroidogenic cells and the Km and V values of the expressed enzyme were determined
M A T E R I A L S A N D M E T H O D S
Animals Adrenal glands and blood were obtained from normal adult Cape baboons Baboon adrenal tissue was obtained from the animal units housed at the University of Cape Town Medical School and the University of Stellenbosch Medical School All anaesthetic and surgical procedures were approved by the Animal Research and Ethics committee
of the two Universities and complied with the Principles of Laboratory Care and the NIH Guide for the Care and Use
of Laboratory Animals 1996 Adrenal glands, allocated for RNA isolation, were flash frozen and stored in liquid N2 For all experiments material was collected from 20 groups consistingof 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
[3H]PROG, [3H]17-OHPROG, DHEA and A4 were pur-chased from Amersham Life Science (Amersham, Bucks, UK) and [3H]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 andb5 contents of baboon adrenal microsomes Microsomes were prepared from baboon adrenal cortex homogenate using standard differential centrifugation tech-niques [19] The cytochrome P450 and cytochrome b5 content of baboon adrenal microsomes were determined as previously described [20] An extinction coefficient of
100 cm)1ÆmM )1 was used for the determination of the cytochrome P450 content while an extinction coefficient of
185 cm)1ÆmM )1was used to determine the concentration of the cytochrome b5[20,21]
Assay for steroid metabolism in baboon adrenal microsomes
The metabolism of PROG and 17-OHPROG was assayed
as previously described [22] Adrenal microsomes (0.5 lM P450) were pre incubated with [3H]PROG and PROG
Trang 3(10 lM) in a total volume of 0.5 mL for 5 min at 37C 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 dichlorodichloro-methane phase was
evaporated under N2 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 WISPTMautomatic injector (Waters) and a
Flo-One liquid scintillation spectrophotometer (Radiomatic,
Tampa, FL) PROG metabolites were separated on a
Novapak C18column 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
100% A to 100% B in 10 min and an isocratic elution with
solvent B for 10 min PREG and 17OH-PREG metabolites
were separated on a Novapak C8column at a flow rate of
1 mLÆmin)1 The mobile phase consisted of solvent A
(water/acetonitrile/isopropanol, 50 : 48.5 : 1.5, v/v/v) and
solvent B (100% acetonitrile) The column was eluted for
5 min with solvent A, followed by a linear gradient from
100% A to 100% B in 2 min and an isocratic elution with
solvent B for 3 min
RNA isolation and reverse transcriptase-polymerase
chain reaction (RT-PCR)
Total RNA was extracted from baboon adrenal cortex with
guanidinium thiocyanate followed by centrifugation in a
cesium chloride solution [23] Polyadenylated RNA (poly
A+) RNA was isolated usinga mRNA Capture kit
(Boehringer Mannheim Biochemicals) Complementary
cDNA was synthesized by reverse transcription of mRNA
usingthe TitanTMOne Tube RT-PCR system (Boehringer
Mannheim Biochemicals) The reverse transcription
reac-tion was performed at 50C for 30 min after which
thermocyclingwas carried out directly Baboon specific
primers, complementary to the 3¢- and 5¢-termini of baboon
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 XhoI and SmaI Nucleotide sequences of both strands, purified RT-PCR product and cloned cDNA, were determined usingthe BigdyeTMVersion
2 diterminator sequencingkit (model 373 A ABI, Applied Biosystems, Foster City, CA)
Assay of CYP17 enzyme activity inHEK-293 cells HEK-293 cells, grown in Dulbecco’s modified Eagle’s medium (DMEM), containing0.9 gÆL)1 glucose, 0.12% NaHCO3, 10% fetal bovine serum, 1% penicillin-strepto-mycin were transfected with the pCI-neo/baboon CYP17 construct, 5 lgÆmL)1, usingdiethylaminoethyl-dextran, 0.25 mgÆmL)1, with the later addition of 100 lM chloro-quine [24] The same protocol was followed to determine the catalytic activity of the recombinant human enzyme, using pcD CYP17 [24] Control transfection reactions were carried out with the plasmid vector pCIneo After 72 h steroidogenic precursors were added to the cells with the appropriate tritium-labelled steroid substrate, [3H]PROG and [3H]PREG Aliquots of 0.5 mL, were removed at specific time intervals and the steroid metabolites were extracted with dichloromethane and analysed by HPLC as described above Immediately after the completion of each experiment, the cells were washed with NaCl/Pi, collected in the same buffer and homogenized with a small glass homogeniser The protein content of the homogenate was subsequently determined by the Bradford method [25]
R E S U L T S
Concentrations of cytochrome P450 and cytochrome
b5in baboon adrenal microsomes The dithionite reduced carbon monoxide vs oxidized difference spectrum of baboon adrenal microsomal cyto-chrome P450 is given in Fig 1A The peak at 425 nm, indicative of cytochrome b5, is reduced upon the addition of 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 b5[20] The concentration of baboon adrenal cytochrome P450 was 0.55 nmolÆmg)1microsomal protein and the concentration of cytochrome b5 was 0.17 nmolÆmg)1protein
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
Trang 4CYP17 activity in adrenal microsomes
A progression curve for the metabolism of PROG by a
baboon adrenal microsomal preparation is shown in Fig 2
At 2 min 65% of the PROG had been metabolized, yielding
40% 17-OHPROG and 10% each of deoxycortisol and
DOC After 12 min the PROG was depleted with
deoxy-cortisol accounted for more than 50% of the radiolabelled
metabolites DOC accounted for 27.5% of the metabolites
indicatingthat the 17a-hydroxylase activity was
consider-ably higher than the 21-hydroxylase activity Typical HPLC
analyses of PROG metabolites present in the medium at 4
and 15 min, respectively, are shown in Fig 3A and B A4 and 16a-hydroxyprogesterone were not detected The metabolism of 17-OHPROG by baboon adrenal micro-somes (Fig 4) showed that at 5 min 50% of the 17-OHPROG was converted to deoxycortisol, the only product, no A4 was detected in the medium
Characterization of baboon CYP17 cDNA RT-PCR amplification of baboon mRNA, usingbaboon specific primers complementary to the nucleotide sequence encodingthe amino and carboxy terminal of baboon
Fig 2 Metabolism of PROG (10 l M ) by baboon adrenal microsomes
(0.5 l M P450).
Fig 3 HPLC analyses of products of PROG
metabolism (10 l M ) by baboon adrenal
microsomes at 4min (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).
Trang 5CYP17 (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 resultingin 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
charged residue H391 corresponding to E in the human
sequence
CYP17 activity in HEK-293 cells transfected
with pCI-neo/baboon CYP17 cDNA
The activity of baboon CYP17 was determined in HEK-293
cells transfected with pCI-neo/baboon CYP17 cDNA
Expression of the recombinant enzyme permitted the
investigation of the catalytic activity for PREG and PROG
away from the competitive influence of 3b-HSD and
CYP21 Metabolism of PREG and PROG by human
CYP17 was also determined to allow comparison of human
and baboon CYP17 in the same HEK-293 cellular
envi-ronment
The conversion of PREG by baboon CYP17 expressed in
HEK-293 cells yielded 17-OHPREG and DHEA (Fig 5)
Initially no DHEA was detected prior to 50% of the PREG
beingconverted to the 17-hydroxy intermediate After 13 h
more than 90% of the PREG was metabolized,
17-OHPREG and DHEA beingthe major metabolites
The conversion of PREG to 17-OHPREG and DHEA by
human CYP17 (Fig 6) proceeded at a similar conversion
rate although the initial accumulation of 17-OHPREG
before DHEA formation was observed, was not as
pronounced as that seen for the conversion of PREG by
baboon CYP17 A comparison of the ratios of
DHEA:17-OHPREG formation duringPREG metabolism
indicated that the biosynthesis of DHEA was initially
slower for baboon CYP17 than for the human enzyme
(Fig 7) The Km and V values for PREG utilization by
baboon CYP17 were 0.9 lMand 0.45 nmolÆh)1Æmg)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 Km
for PROG utilization by baboon CYP17 expressed in
HEK-293 cells, was 6.5 lMand the maximum velocity (V value)
was 3.9 nmolÆh)1Æmg)1protein (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
HEK-293 cells expressinghuman CYP17
Fig 5 Time course of PREG (1 l M ) metabolism by baboon CYP17 expressed in HEK-293 cells.
Fig 6 Time course of PREG (1 l M ) metabolism by human CYP17 expressed in HEK-293 cells.
Trang 6The Kmand V values for PROG and PREG utilization
by the expressed cytochromes CYP17, are summarized in
Table 1
D I S C U S S I O N
Cytochromes CYP17 from various species display distinctly
different catalytic activities, unique to their physiological
requirements These differences, in which specific metabolic
routes in the steroidogenic pathway are favoured, yielding
different levels of steroid production, can be attributed to
the species-specific hydroxylase and lyase activities of
CYP17 for PREG, PROG and the hydroxylated
interme-diates The expression of the recombinant enzyme from
various species in nonsteroidogenic systems has led to the
characterization of CYP17 at enzymatic and molecular
levels [26,27] In the absence of a crystal structure these
investigations have contributed to a better understanding of
structure/function relationships and comparative analyses between different species have identified specific domains 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 pertainingto 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.
Species K m (l M ) V (nmolÆh)1Æmgprotein)1) K m (l M ) V (nmolÆh)1Æmgprotein)1)
Fig 7 Ratio of 17-OHPREG and DHEA formation during PREG
(1 l M ) metabolism by baboon and human CYP17 expressed in HEK-293
cells.
Fig 8 Kinetics of PREG metabolism by baboon CYP17 expressed in HEK-293 cells Apparent K m ¼ 0.9 l M ; V value ¼ 0.45 nmolÆh)1Æmg)1protein Results are representative of at least three independent experiments.
Trang 7similarities to human CYP17 in the catalytic activity,
makingthe baboon an important CYP17 candidate for
the study of structure/function relationships
CYP17 is a membrane-bound microsomal cytochrome
P450 In the adrenal gland the activity of this enzyme is
influenced, not only by the cellular lipid environment, but
also by the presence of electron transport proteins,
cytochrome P450 reductase and cytochrome b5 In
addi-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 duringthe metabolism of PROG
by baboon CYP17 A4 was also not detected as a product
of PROG metabolism indicatingthat baboon CYP17, like
human CYP17, has little, if any, lyase activity towards
17-OHPROG Cytochrome b5, a modulatingagent of
CYP17 activity, was present in the baboon adrenal
Fig 9 Time course of PROG (1 l M ) metabolism by baboon CYP17
expressed in HEK-293 cells Insert: HPLC analyses of PROG
meta-bolism (1 l M ) by baboon CYP17 expressed in HEK-293 cells Peaks
on the chromatogram are: 1, PROG (20.5 min); 2, 17-OHPROG
(17.25 min) No 16-OHPROG or A4 was detected.
Fig 10 Time course of PROG (1 l M ) metabolism by human CYP17 expressed in HEK-293 cells.
Fig 11 Kinetics of PROG metabolism by baboon CYP17 expressed in HEK-293 cells (apparent K m ¼ 6.5 l M ; V value ¼ 3.9 nmolÆh)1Æmg)1 protein) Results are representative of at least three independent experiments.
Trang 8microsomal preparations and the ratio of cytochrome
P450 to cytochrome b5 was 3 : 1
A comparison between the microsomal baboon CYP17
investigated in this study and human microsomal CYP17
previously reported [1], suggested similarities as well as
differences in the functional activities of the enzyme in these
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
encodingbaboon 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 Kmand 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
this aspect needs to be studied further as it has an important
bearingon the ability of the baboon to produce adrenal
C19-steroids, particularly if the baboon is producinghigh
levels of cortisol
In contrast to PREG metabolism, baboon CYP17
expressed in HEK-293 cells converted PROG to
17-OHPROG much faster than the human enzyme
(Table 1) The higher V value obtained for the baboon
enzyme could be attributed to a higher expression level than
the human enzyme due to differences in expression vectors
used It is, however, not apparent how differences in the
expression levels could result in the differences observed
in the apparent Kmvalues obtained (Table 1) In addition
it is important to note that the same differences were
not observed for the metabolism of PREG by the two
enzyme preparations Despite the large degree of homology
between the human and baboon CYP17 the expressed
baboon CYP17 was unable to catalyse the formation of
16a-hydroxyprogesterone Both expressed enzymes had no
lyase activity towards PROG or 17-OHPROG
Understandingof the complexity of hydroxylase and
lyase activity of CYP17 by way of primary sequence
alignments of CYP17 from different species has limitations
Even though CYP17 homology ranges from 65 to 78%,
makingdeductions pertainingto structure/function
rela-tionships, usinginterspecies primary sequence alignments,
has been hampered due to the variation in catalytic activities
amongst species A combination of computational
model-lingand 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 dockingand binding, the active site includingthe heme-bindingdomain and redox partner bindingdomain [10] Baboon and human CYP17 are excellent candidates for identifyingregions 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 Kmfor PROG and no 16a-hydroxylase activity
Sequence alignments of CYP17 based on the structures of bacterial cytochromes P450 (accordingto 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 between the three species, i.e baboon KIVHN, human NVIQN and guinea pig VTIRR Baboon and guinea pig have two positively charged residues whereas human has uncharged polar residues Site directed mutagenesis would show if the increased activity towards PROG observed in the expressed enzyme, could be attributed to these differ-ences Furthermore, it is possible that baboon CYP17, with the high degree of homology and distinct catalytic differ-ences to human CYP17, would permit a study of the role of specific domains in the structure/function relationship of CYP17
A C K N O W L E D G E M E N T S
The authors wish to acknowledge the support of the National Research Foundation; Sandy Graham for fruitful discussions and Bjarne Faurholm for his technical assistance Human pCD CYP17 was a kind gift from Prof R.W Estabrook.
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