Cytochrome P-450terp. Isolation and purification of the protein and cloning and sequencing of its operon.

Cytochromes P-450 are extremely important in the oxidative metabolism of a variety of endogenous and exogenous compounds in pro- and eukaryotic organisms. Progress in understanding the structure and mechanism of action of this superfamily of enzymes has been hampered by the properties of the eukaryotic enzymes and the availability of only one well-characterized prokaryotic enzyme as a model. We report here the isolation of a Pseudomonas species which will utilize a monoterpene natural product, alpha-terpineol, as its sole source of carbon and energy. Approximately 1% of the soluble protein in the cell-free extract is a novel cytochrome P-450 (P-450terp). This enzyme and its associated iron sulfur protein electron carrier (terpredoxin) have been purified to homogeneity and their NH2-terminal amino acid sequences determined. The amino acid sequences of six tryptic peptide fragments of cytochrome P-450terp have also been determined. This sequence information was used to clone the gene encoding cytochrome P-450terp. Three clones representing approximately 8 kilobase pairs of unique sequences were selected and sequenced. Five non-overlapping open reading frames (ORFs) were found in the sequences, and the translated sequences were used to search the Protein Identification Resource for comparable proteins. The ORFs were identified as: 1) an alcohol dehydrogenase, 2) an aldehyde dehydrogenase, 3) cytochrome P-450terp, 4) terpredoxin reductase, and 5) terpredoxin. The identification of both the cytochrome P-450terp and terpredoxin DNA sequence was confirmed by the presence of each of the corresponding amino acid sequences found in the purified proteins. The five ORFs were bounded on both the 5' and 3' ends by consensus factor-independent terminator sequences. A consensus promoter sequence was found immediately 5' to the first ORF. These results indicate that we have sequenced the complete terp operon. Comparison of the amino acid sequence of cytochrome P-450terp to that of all other cytochromes P-450 has shown that it is the first member of the gene family CYP108. Preliminary characterization of the chemical and physical properties and the preparation of crystals of this new cytochrome P-450, suitable for x-ray diffraction analysis, indicate that it will be useful in comparison studies with other members of this class of proteins.

Nucleotide sequence of a cDNA encoding porcine testis 17 alpha-hydroxylase cytochrome P-450.

We describe the isolation and characterization of a cDNA encoding the complete porcine neonatal testis 17 alpha-hydroxylase/C-17,20-lyase cytochrome P-450. The deduced amino acid sequence is 509 amino acids in length.

3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase expression in rat and characterization of the testis isoform.

The isolation, cloning and expression of a DNA insert complementary to mRNA encoding rat testis 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta-HSD) is reported. The insert contains an open reading frame encoding a protein of 373 amino acids, which exhibits 73% and 78% identity to the cDNA encoding the human placental form at the amino acid and nucleotide levels respectively. Northern blot analysis of total RNA of rat tissues using as probe a specific radiolabeled cDNA insert encoding rat testis 3 beta-HSD demonstrated high levels of 1.6 kb mRNA species in ovary, adrenal and Leydig tumor, with lower but detectable message in testis and adult male liver, while the probe also hybridized to a 2.1 kb mRNA species in liver. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to 3 beta-HSD present in H540 Leydig tumor cell homogenate and human placental microsomal 3 beta-HSD, as detected by immunoblot analysis, and catalyzed the conversion of pregnenolone to progesterone, 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, but not NADP+, converted pregnenolone to progesterone and dehydroepiandrosterone to androstenedione with apparent Km values of 0.13 and 0.09 microM, respectively. Immunoblot analysis of various rat tissues using a polyclonal antibody directed against human placental 3 beta-HSD, in addition to immunoreactivity in the adrenal and testis, demonstrated immunoreactive 3 beta-HSD protein in adult male liver, but not in adult female or fetal liver. We conclude that while one gene product is highly expressed in testicular Leydig cells, and probably adrenal and ovary, accounting for their 3 beta-HSD content, a 3 beta-HSD is also expressed in liver in a sex-specific manner.

Structure-function relationships of human aromatase cytochrome P-450 using molecular modeling and site-directed mutagenesis.

The conversion of androgens to estrogens is catalyzed by an enzyme complex named aromatase, which consists of a form of cytochrome P-450, aromatase cytochrome P-450 (cytochrome P-450AROM), and the flavoprotein, NADPH-cytochrome P-450 reductase. As a first step toward investigation of the structure-function relationships of cytochrome P-450AROM, we have used computer modeling to align the amino acid sequence of cytochrome P-450AROM with that of cytochrome P-450CAM from Pseudomonas putida and thus create a substrate pocket using the heme-binding region and the I-helix of cytochrome P-450CAM as the template. Site-directed mutagenesis was then carried out at two sites: one at a region that aligns with the bend in the I-helix of cytochrome P-450CAM and the other at a glutamate (Glu302) just N-terminal of this bend, which is predicted to be in close proximity to the C2-position of the androstenedione substrate. To determine the importance of the former region, three mutants were constructed: A307G (Ala307----Gly), P308V (Pro308----Val), and GAGV, which changed -Ile305-Ala306-Ala307-Pro308- to -Gly-Ala-Gly-Val- (the corresponding sequence found in 17 alpha-hydroxylase cytochrome P-450). When these proteins were expressed in COS-1 cells, it was found that the activity of P308V was approximately one-third that of the wild type. These observations are consistent with the concept that Pro308 causes a bend in the I-helix of cytochrome P-450AROM, similar to that observed in cytochrome P-450CAM, which is believed to be important in forming the substrate-binding pocket. The next set of mutants were designed to determine the importance of Glu302 in catalysis. Four mutants were prepared in which Glu302 was changed either to Ala, Val, Gln, or Asp, and the activities of the expressed proteins were examined. It was found that mutations in which the carboxylic acid was replaced were essentially devoid of activity. On the other hand, changing Glu302 to Asp resulted in a two-thirds reduction in the apparent Vmax. These results support the role of a carboxylic acid residue at position 302 in the catalytic activity of cytochrome P-450AROM.

Expression of mRNA species encoding steroidogenic enzymes in the rat ovary.

We have examined the levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P-450scc), 17 alpha-hydroxylase cytochrome P-450 (P-450(17 alpha), aromatase cytochrome P-450 (P-450AROM) and 3 beta-hydroxysteroid dehydrogenase (3 beta-HSD) in rat ovaries throughout the oestrous cycle, during pregnancy and in immature animals treated with pregnant mare serum gonadotrophin (PMSG). Total or poly(A)(+)-enriched RNA was prepared from adult rat ovaries throughout the oestrous cycle, from immature rat ovaries 24 and 48 h after treatment and from adult rat ovaries on days 10, 14, 17 and 21 of gestation. Expression of the mRNA species was examined by Northern analysis using specific [32P]cDNA probes. During the oestrous cycle P-450scc mRNA of approximately 1.9 kb was detected at low levels, while 3 beta-HSD mRNA of 1.7 kb was in relatively high abundance throughout the oestrous cycle. While P-450(17) alpha mRNA of 1.9 kb and P-450AROM of 2.7, 2.2 and 1.7 kb were highly abundant during dioestrus, pro-oestrus and oestrus, the levels of these mRNA species decreased markedly to be nearly undetectable during metoestrus. During pregnancy there was considerably more variation in the expression of the mRNA species examined. Expression of P-450scc mRNA was at low, but detectable, levels until day 14, thereafter expression increased to high levels (day 14-21 of gestation). Levels of P-450(17) alpha mRNA on day 10 of gestation were lower than at pro-oestrus during the oestrous cycle and decreased further on days 14 and 17. Expression of 3 beta-HSD was decreased on day 10, but on days 14, 17 and 21 of gestation high mRNA levels were detectable. Ovarian expression of the three P-450AROM species was dramatically increased between days 14 and 17 of pregnancy, but declined by day 21. In immature rats, P-450scc mRNA was detected at low levels in unstimulated animals and increased markedly after treatment with PMSG, while subsequent treatment with human chorionic gonadotrophin (hCG) had a minimal effect on expression. Expression of P-450(17) alpha mRNA was high in unstimulated immature and PMSG-treated rats, but diminished after treatment with hCG. All three P-450AROM mRNA species were undetectable in ovaries from unstimulated immature animals; however, induction of all three was observed in PMSG-treated rats, but this expression decreased to undetectable levels upon subsequent administration of hCG.(ABSTRACT TRUNCATED AT 400 WORDS)

Characterization of the steroid-metabolizing capacity of the hepatic cytochrome P450IIC5 expressed in COS-1 cells: 3 beta-hydroxysteroid dehydrogenase/delta 5----4 isomerase type activity.

Cytochrome P450IIC5 (rabbit liver 21-hydroxylase) is unusual among hepatic forms of cytochromes P450 because it catalyzes the conversion of one active steroid hormone (progesterone) to another active hormone (deoxycorticosterone). Another interesting aspect of this steroid-hydroxylating enzyme is the ability to convert delta 5-3 beta-hydroxysteroids to the delta 4-3-ketosteroid configuration. The delta 5-3-beta-hydroxysteroid, pregnenolone, was readily 21-hydroxylated, and this product was further metabolized to the delta 4-3-ketosteroid, deoxycorticosterone. It is suggested that the mechanism of this cytochrome P450-mediated, 3 beta-hydroxysteroid dehydrogenase/delta 5----4 isomerase-like reaction is through a gem-diol formation. In this study, COS-1 cells were transfected with the plasmid encoding cytochrome P450IIC5 to express a functional enzyme within the cell milieu. Transfected COS cells preferentially metabolize pregnenolone compared with all other steroids tested. Progesterone and 17 alpha-hydroxypregnenolone are also 21-hydroxylated, whereas 17 alpha-hydroxyprogesterone is a poor substrate. Substrate preference of this 21-hydroxylase differs from that seen with bovine adrenal P450XXIA1 (formerly P450C21) hydroxylase. Additionally, this study demonstrated that C19 steroids, like dehydroepiandrosterone and androstenedione, are hydroxylated at the 16 alpha position. Contrary to previous reports, no metabolite of estradiol-17 beta was detected, presumably due to the unstable nature of catechol estrogens (2-hydroxyestradiol).

Expression of cytochrome P-450(17) alpha, 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase, and steroid 5 alpha-reductase in rat H540 Leydig tumor cells.

The rat H540 Leydig tumor cell is established as a model for acute lutropin action on the initial step of steroidogenesis, namely the conversion of cholesterol to pregnenolone. Herein, we demonstrate that H540 cells express high levels of three steroid-metabolizing enzymes which are involved in the further processing of pregnenolone in the endoplasmic reticulum of the steroidogenic cell. In particular, in addition to expressing 17 alpha-hydroxylase cytochrome P-450 (P-450(17) alpha) and 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta-HSD), H540 cells also showed high levels of steroid 5 alpha-reductase mRNA and activity. The H540 cells therefore exhibit similarity to Leydig cells from sexually immature animals which also demonstrate high 5 alpha-reductase activity. Thus, after 3 beta-HSD-catalyzed formation from pregnenolone, progesterone was efficiently converted to 5 alpha-pregnan-3,20-dione (5 alpha-dihydroprogesterone) and subsequent metabolism to the corresponding 17 alpha-hydroxylated derivative and 5 alpha-androstan-3,17-dione in a reaction catalyzed by P-450(17) alpha. H540 cells have apparently very low 17-ketosteroid reductase activity and, therefore, a principal end-product of the steroidogenic pathway in these cells was 5 alpha-androstan-3,17-dione. H540 cells maintained in primary culture under serum-free conditions accumulated demonstrable levels of mRNA species for P-540 17 alpha (1.7 kb), 3 beta-HSD (1.6 kb) and 5 alpha-reductase (2.7 kb). This finding suggests that the H540 tumor cell model will not only be of utility in the study of acute lutropin action but also in the elucidation of mechanisms involved in the regulation of expression of various families of microsomal steroid-metabolizing enzymes.

Structural analysis of the gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase.

The structural gene encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4-isomerase (3 beta HSD) was isolated from a human EMBL3 genomic library. The gene encompasses approximately 8 kilobases of DNA and is comprised of two large introns and three exons encoding amino acid residues 1-48, 49-103, and 104-373, respectively. The exonic sequence is identical to that of the cDNA that we previously isolated and expressed in COS 1 cells. DNA sequence analysis reveals a putative TATA (TATATAA) motif 26 basepairs up-stream of the beginning of exon I, as determined by S1 nuclease protection analysis. However, primer extension analysis using poly(A)+ RNA isolated from both placenta and corpora lutea indicates that the RNA initiates up-stream of the putative TATA motif, and that an additional 53-basepair exon, which is untranslated, is present 5' to the first coding exon. Southern hybridization analysis of genomic DNA using a single exon probe suggests that there may be more than one copy of the gene in the human genome. In addition, we confirm from Southern analysis of genomic DNA isolated from human x hamster somatic cell hybrids that the gene is located on human chromosome 1. These findings will provide a foundation for the characterization of apparent 3 beta HSD clinical deficiencies when these are due to a mutation in the structural gene.

Physical analysis of phr gene transcription in Escherichia coli K-12.

The phr gene of Escherichia coli K-12 encodes the light-dependent, DNA repair enzyme photolyase, which removes UV light-induced pyrimidine dimers from cellular DNA. From Southern hybridization analysis of several strains containing successively extended phr deletions, we have determined the direction of transcription of the phr gene on the E. coli K-12 chromosome. Northern (RNA) hybridization analysis suggests that the phr gene is cotranscribed with a previously identified gene of unknown function (orf169) into two messages of different lengths. S1 nuclease mapping analysis indicates that the two transcripts share a single termination site but initiate at two different sites. Finally, we have determined that the presence of orf169 is not necessary for phr gene activity in vivo.

Construction and expression of human/bovine P45017 alpha chimeric proteins: evidence for distinct tertiary structures in the same P450 from two different species.

In the human and bovine adrenal cortex, 17 alpha-hydroxylase (P45017 alpha) catalyzes reactions involved in the production of C21-glucocorticoids (17 alpha-hydroxylation) and C19-androgens (17,20-lyase). The bovine and human forms of P45017 alpha share 71% primary sequence identity. Using naturally occurring restriction sites common to cDNAs encoding both human and bovine P45017 alpha, we have constructed bovine/human (bovine amino terminus and human carboxy terminus) and human/bovine (human amino terminus and bovine carboxy terminus) cDNAs that have been expressed in COS 1 cells, and the enzymatic properties of the resultant chimeric proteins have been examined. The three bovine/human chimeras studied have 17 alpha-hydroxylase activities intermediate between those of the wild-type bovine and wild-type human enzymes, although the 17,20-lyase activity of these chimeras is significantly lower than that of either of the wild-type enzymes. Surprisingly, the opposite chimeras (those containing a human amino-terminal sequene and a bovine carboxy-terminal sequence) are all virtually inactive, even though they appear to be expressed at normal levels. These results indicate that the folding of P45017 alpha initiated by the bovine amino terminus can accommodate human P45017 alpha sequences of various lengths to produce a relatively normal 17 alpha-hydroxylase having decreased 17,20-lyase activity. On the other hand, folding initiated by the human P45017 alpha amino terminus does not easily accommodate bovine carboxy-terminal sequences to produce a functional enzyme. Presumably this difference arises from the fact that the tertiary structures of the bovine and human forms of P45017 alpha are sufficiently different so that interchanging sequences will not lead to functional enzymes in a predictable fashion.

Human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase from placenta: expression in nonsteroidogenic cells of a protein that catalyzes the dehydrogenation/isomerization of C21 and C19 steroids.

The isolation, cloning, and expression of a cDNA insert complementary to mRNA encoding human 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase is reported. The insert contains an open reading frame encoding a protein of 372 amino acids, the initial 29 amino acids corresponding to the N-terminal sequence identified from the purified human placental microsomal enzyme. The cDNA was inserted into a modified pCMV vector and expressed in COS-1 monkey kidney tumor cells. The expressed protein was similar in size to human placental microsomal 3 beta-hydroxysteroid dehydrogenase/delta 5----4isomerase, as detected by immunoblot analysis, and catalyzed the conversion of 17 alpha-hydroxypregnenolone to 17 alpha-hydroxyprogesterone, pregnenolone to progesterone, and dehydroepiandrosterone to androstenedione. Transfected COS cell homogenates, supplemented with NAD+, very efficiently oxidized 5 alpha-androstan-3 beta,17 beta-diol to 5 alpha-dihydrotestosterone and, upon addition of NADH, reduced 5 alpha-dihydrotestosterone to 5 alpha-androstan-3 beta,17 beta-diol. Thus, the dehydrogenation/isomerization steps of steroid biosynthesis can be catalyzed by a single polypeptide chain, which can metabolize all of the major physiological substrates.

Putidaredoxin reductase and putidaredoxin. Cloning, sequence determination, and heterologous expression of the proteins.

The oxidation of camphor by cytochrome P-450cam requires the participation of a flavoprotein, putidaredoxin reductase, and an iron-sulfur protein, putidaredoxin, to mediate the transfer of electrons from NADH to P-450 for oxygen activation. A 2.2-kilobase pair BamHI-StuI fragment from whole cell DNA of camphor-grown Pseudomonas putida has been cloned and sequenced. Translation of the sequence revealed two open reading frames that could code for putidaredoxin reductase and putidaredoxin. In the case of putidaredoxin, the translated sequence matched the published sequence (Tanaka, M., Haniu, M., Yasunobu, K. T., Dus, K., and Gunsalus, I. C. (1974) J. Biol. Chem. 249, 3689-3701) with the exception of one amino acid. Codon usage in these proteins, like the proteins of other Pseudomonads, is strongly biased to G + C in the third nucleotide. A potential transcription termination site was found 3' to the putidaredoxin coding region. The "FAD-binding" amino acid consensus sequence, present in other flavoproteins, was found in putidaredoxin reductase beginning at residue 11 and a second occurrence of this sequence was found beginning with amino acid 156. The second sequence could represent the NAD-binding site. The regions encoding putidaredoxin reductase and putidaredoxin were subcloned and independently expressed in Escherichia coli at the level of 0.4 and 4.8 mg of enzymatically active protein/g wet weight of cells, respectively. Site-directed mutagenesis was used to change the rare start codon, GTG, of putidaredoxin reductase to ATG which resulted in an 18-fold increase in the level of expression of this protein to 7.4 mg/g wet weight of cells. The construction of these two clones, which express these important proteins, will facilitate studies of their interaction with each other and with P-450cam.

Expression of messenger ribonucleic acid species encoding steroidogenic enzymes in human follicles and corpora lutea throughout the menstrual cycle.

The levels of expression of mRNA species encoding cholesterol side-chain cleavage cytochrome P-450 (P450scc), 17 alpha-hydroxylase cytochrome P450 (P450(17 alpha], aromatase cytochrome P-450 (P-450AROM), and 3 beta-hydroxysteroid dehydrogenase (3 beta HSD) were examined in human follicles and corpora lutea (CL) throughout the menstrual cycle. Tissues were obtained from women undergoing hysterectomy and oophorectomy. The largest follicle or the CL was dissected from the ovary depending on whether the surgery was performed in the follicular or luteal phase. The day of the cycle was determined by onset of last menstrual period and was confirmed by endometrial histology. Total RNA was examined by Northern blot analysis, using as probes specific 32P-labeled cDNA inserts encoding each human enzyme. Early follicles demonstrated detectable mRNA for both P450scc and P450(17 alpha), but not for P450AROM or 3 beta HSD. P450AROM was detectable late in the follicular phase and appeared markedly induced in the CL. 3 beta HSD was detectable only in the CL. Levels of P450(17 alpha) mRNA remained relatively unchanged throughout the cycle, whereas P450scc mRNA levels were greatly increased in the CL. The presence of P450(17 alpha) mRNA in the human CL is of interest, since it is absent from the bovine CL, and this is consistent with the ability of the human, but not the bovine, CL to synthesize 17 alpha-hydroxyprogesterone and estrogens. The fact that P450AROM expression is highest in CL is surprising, since plasma estrogen levels are highest during the late follicular phase of the cycle, and may suggest that CL estrogen biosynthesis is limited by 17 alpha-hydroxylase or 17,20-lyase activities.

Expression of a full-length cDNA encoding bovine adrenal cytochrome P450C21.

Two full-length cDNA clones encoding bovine adrenocortical P450C21 have been constructed in a eukaryotic expression vector using partial-length cDNAs whose structures have been previously reported. Following expression of these cDNAs in COS 1 cells, the substrate specificity of P450C21 was determined. Of the 18 steroids tested, progesterone, 17 alpha-hydroxyprogesterone, and 11 beta,17 alpha-dihydroxyprogesterone were found to be the only steroids to serve as substrates for this adrenal enzyme, a much higher degree of substrate specificity than has been reported for a hepatic 21-hydroxylase. The Vmax for 17 alpha-hydroxyprogesterone was 2.5 times greater than that for progesterone, whereas delta 5-steroids were unable to serve as substrate for this enzyme. A difference between the two cDNAs is located at amino acid 401 where one resultant enzyme contains tyrosine while the other contains histidine. This amino acid difference appears to have no effect on the kinetic properties of adrenal P450C21.

Rat P450(17 alpha) from testis: characterization of a full-length cDNA encoding a unique steroid hydroxylase capable of catalyzing both delta 4- and delta 5-steroid-17,20-lyase reactions.

A cDNA clone encoding the complete rat 17 alpha-hydroxylase (P450(17 alpha] from testis has been identified and sequenced. The deduced amino acid sequence is found to have 69% similarity with human P450(17 alpha), 64% similarity with bovine P450(17 alpha), and 47% similarity with chicken P450(17 alpha). The protein contains 507 amino acids being one amino acid shorter than the human P450(17 alpha) as the result of a codon being absent at the position of amino acid 139 in the human sequence. The cDNA hybridizes to a single mRNA (approximately 2.0 kilobases) in rat testis RNA and Southern analysis indicates the presence of a single CYP17 gene in the rat genome. Expression of this cDNA in COS1 cells leads to production of a steroid hydroxylase which is capable of converting both 17 alpha-hydroxypregnenolone and 17 alpha-hydroxyprogesterone into C19 steroids, dehydroepiandrosterone, and androstenedione, respectively. This activity profile is distinct from that of either the human or bovine forms of P450(17 alpha) which are unable to catalyze 17,20-lyase conversion of delta 4-C21 steroids to delta 4-C19 steroids at significant rates.

Sequences of the Escherichia coli photolyase gene and protein.

We have determined the nucleotide sequence of a 2039-base pair segment of Escherichia coli chromosomal DNA containing the phr gene, which encodes deoxyribopyrimidine photolyase. The coding region of phr is 1416 base pairs and is preceded by regions homologous to consensus sequences for E. coli promoters and ribosome binding sites. The phr gene is preceeded by an open reading frame of 169 codons (orf169) which is transcribed in the same direction. The proximity of orf169 to phr suggests that both are members of a single operon containing one or more internal promoters allowing differential expression of phr. An unusually large number of rare or infrequently used codons are utilized in phr, which may contribute to the low copy number of photolyase. The sequence at the NH2 and COOH termini and the overall amino acid composition of mature photolyase, determined using purified protein, agrees with predictions based upon the nucleotide sequence. Photolyase consists of 471 amino acids and has a calculated molecular weight of 53,994.

Convenient construction of recA deletion derivatives of Escherichia coli.

Two Escherichia coli K-12 Hfr strains have been constructed which transfer a recA deletion, which is highly linked to a Tn10 insertion conferring tetracycline resistance, early during conjugation. These strains transfer the recA deletion in opposite directions with different origins of transfer, allowing for preservation of desirable recipient strain markers either clockwise or counterclockwise of recA.