Báo cáo Y học: Baboon cytochrome P450 17a-hydroxylase/17,20-lyase (CYP17) Characterization of the adrenal microsomal and cloned enzymes docx

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 cells. A major difference between species resides in the lyase activity towards the hydroxylated intermediates and in the fact that the secretion of C 19 -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 K m and V values for pregnenolone and progesterone of 0.9 l M and 0.4 nmolÆh )1 Æmg protein )1 and 6.5 l M and 3.9 nmolÆh )1 Æmg protein )1 , respectively. Baboon CYP17had 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 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. The 17,20-lyase reaction cleaves the C17,20 bond converting these hydroxylated intermediates to dehydroepiandroster- one (DHEA) or androstenedione (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 C 19 -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 C 19 -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 oxidation reactions 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 Bag X1, 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-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]. 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, including man. 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 resulting in the hypersecre- tion of ACTH and an increased biosynthesis of deoxycor- ticosterone (DOC) and corticosterone. 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%, resulting in the prediction of domains playing a catalytic role rather than specific amino acids. The hydroxylase activity of CYP17 for the D 5 -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 human CYP17 with a species much more closely related than investigated thus far, and may contribute to a further understanding of the hydroxylase and lyase activities of the enzyme in relation to substrate binding and 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- ing CYP21 enzyme and of membrane components including NADPH-cytochrome P450 reductase, cytochrome b 5 and phospholipids. The gene encoding baboon CYP17 was isolated from baboon adrenal mRNA. The recombinant DNA was expressed in nonsteroidogenic cells and the K m and V values of the expressed enzyme were determined. MATERIALS AND METHODS 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 ÔPrinciples of Laboratory CareÕ and the ÔNIH Guide for the Care and Use of Laboratory AnimalsÕ 1996. Adrenal glands, allocated for RNA isolation, were flash frozen and stored in liquid N 2 . 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 homogenate using standard differential centrifugation tech- niques [19]. The cytochrome P450 and cytochrome b 5 content of baboon adrenal microsomes were determined as previously described [20]. An extinction coefficient of 100 cm )1 Æm M )1 was used for the determination of the cytochrome P450 content while an extinction coefficient of 185 cm )1 Æm M )1 was used to determine the concentration of the cytochrome b 5 [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 l M P450) were pre incubated with [ 3 H]PROG and PROG Ó FEBS 2002 Characterization of baboon CYP17 (Eur. J. Biochem. 269) 5609 (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 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 NovapakÒ C 8 column at a flow rate of 1mLÆmin )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 using a mRNA Capture kit (Boehringer Mannheim Biochemicals). Complementary cDNA was synthesized by reverse transcription of mRNA using the Titan TM One Tube RT-PCR system (Boehringer Mannheim Biochemicals). The reverse transcription reac- tion was performed at 50 °Cfor30minafterwhich thermocycling was carried out directly. Baboon specific primers, complementary to the 3¢-and5¢-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 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 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, [ 3 H]PROG and [ 3 H]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/P i , 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]. RESULTS Concentrations of cytochrome P450 and cytochrome b 5 in 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 b 5 , 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 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 . 5610 A. C. Swart et al. (Eur. J. Biochem. 269) Ó FEBS 2002 CYP17 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 indicating that 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, using baboon specific primers complementary to the nucleotide sequence encoding the 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 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 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 being converted to the 17-hydroxy intermediate. After 13 h more than 90% of the PREG was metabolized, 17-OHPREG and DHEA being the 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 during PREG metabolism indicated that the biosynthesis of DHEA was initially slower for baboon CYP17 than for the human enzyme. (Fig. 7). The K m 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 HEK-293 cells expressing human 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. 5612 A. C. Swart et al. (Eur. J. Biochem. 269) Ó FEBS 2002 The K m and V values for PROG and PREG utilization by the expressed cytochromes CYP17, are summarized in Table 1. DISCUSSION 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 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 ) V (nmolÆh )1 Æmg protein )1 ) Baboon 6.5 ± 0.1 3.9 0.9 ± 0.05 0.45 Human 0.8 ± 0.1 0.5 1.3 ± 0.2 0.5 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 )1 Æmg )1 protein. Results are representative of at least three independent experiments. Ó FEBS 2002 Characterization of baboon CYP17 (Eur. J. Biochem. 269) 5613 similarities to human CYP17 in the catalytic activity, making the 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 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 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 )1 Æmg )1 protein). Results are representative of at least three independent experiments. 5614 A. C. Swart et al. (Eur. J. Biochem. 269) Ó FEBS 2002 microsomal preparations and the ratio of cytochrome P450 to cytochrome b 5 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 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 this aspect needs to be studied further as it has an important bearing on the ability of the baboon to produce adrenal C19-steroids, particularly if the baboon is producing high 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 K m values 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. Understanding of 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%, making deductions pertaining to structure/function rela- tionships, using interspecies primary sequence alignments, has been hampered due to the variation in catalytic activities amongst species. A combination of computational model- 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 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. ACKNOWLEDGEMENTS 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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