Complete amino acid sequence of 21-hydroxylase cytochrome P-450 from porcine adrenal microsomes

Adrenal microsomal 21-hydroxylase is essential for biosynthesis or metabolism of various steroid hormones. The complete amino acid sequence of this cytochrome P-450 from pig adrenal was determined by sequence analysis on several sets of proteolytic fragments. The mature protein consists of 492 amino acid residues, corresponding to a molecular weight of 55,484. Seven out of nine total cysteine residues are localized in the amino terminal half of the molecule. The carboxyl terminal half contains only two cysteines, one of which is located at the highly conserved heme-binding region proposed in all cytochromes P-450. A structural comparison between 21-hydroxylase and 17 alpha-hydroxylase reveals that there is a preponderance of sequence homology at the carboxyl terminal region. These studies indicate that a single gene product is expressed for steroid 21-hydroxylase in porcine adrenal glands.     

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.     

The cDNA and protein sequence of a phenobarbital-induced chicken cytochrome P-450

Several cDNA clones complementary to a chicken phenobarbital-inducible cytochrome P-450 have been isolated and sequenced, representing the first non-mammalian eukaryotic cytochrome P-450 sequence to be analyzed. The cDNA clones hybridized to two mRNAs of 3.5 and 2.5 kilobases in length, but further analysis indicated that the clones were derived from the larger mRNA. The sequence contains a 5'-noncoding region of 39 nucleotides and an open reading frame of 1473 nucleotides. The remainder of the sequence is due to the 3'-noncoding region and poly(A) tail. The open reading frame encodes a protein of 491 amino acids with a molecular weight of 56,196. The chicken cytochrome P-450 shows an overall homology of 45-54% compared with the mammalian phenobarbital-induced cytochrome P-450s. The degree of homology is not uniform, with some short regions showing much greater levels of sequence conservation. In particular, the chicken cytochrome P-450 contains the conserved cysteinyl domain near the carboxyl terminus, found in all cytochrome P-450s and which is thought to be involved in heme binding. Using the chicken sequence, a more accurate estimate of the evolutionary rates of cytochrome P-450s has been made. It is suggested that the phenobarbital-, 3-methylcholanthrene, and pregnenolone 16 alpha-carbonitrile-induced cytochrome P-450 gene families diverged from a common ancestral gene 600 million years ago. Furthermore the phenobarbital-inducible gene apparently underwent gene duplication events at about the time of the divergence of the chicken and mammalian lineages. The results imply that most mammals should have at least four rather distantly related phenobarbital-inducible gene subfamilies.     

Multiple gene-like sequences related to the rabbit hepatic progesterone 21-hydroxylase cytochrome P-450 1

Rabbits exhibit phenotypic differences, 21H and 21L, in the rate of hepatic progesterone 21-hydroxylation that reflect 10-fold higher microsomal concentrations of cytochrome P-450 1 in 21H rabbits. A cDNA library in pBR322 was prepared from liver mRNA isolated from a 21H rabbit. A clone, p1-8, producing a hybrid protein resulting from the insertion of the cDNA into the beta-lactamase gene of the plasmid expressed 5 distinct epitopes that were recognized by a panel of monoclonal antibodies developed toward P-450 1. RNAs selected from total hepatic mRNA by filter hybridization with p1-8 yield at least two electrophoretically distinct proteins when translated in vitro and immunoprecipitated with the 3C3 monoclonal antibody. Only one of the two proteins is recognized by the 1F11 monoclonal antibody, which is highly specific for P-450 1, and the immunoprecipitated protein exhibits the electrophoretic mobility of P-450 1. The other protein remains unidentified. Northern blot analysis indicates that the 3' noncoding portion of p1-8 hybridizes to higher steady state concentrations of polyadenylated RNA in the 21H as compared to 21L rabbits. This correspondence in expression with that of P-450 1 in the 21H and 21L phenotypes further suggests that p1-8 encodes P-450 1 or a closely related protein. The cDNA is 1871 base pairs in length and encodes a protein of 487 amino acids. Southern blot analysis indicates that several independent, gene-like sequences hybridize with the 3' noncoding region of p1-8 under conditions of high stringency. These results indicate that P-450 1 is a member of an extensive multigene family.     

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.     

Why human cytochrome P450c21 is a progesterone 21-hydroxylase

Human cytochrome P450c21 (steroid 21-hydroxylase, CYP21A2) catalyzes the 21-hydroxylation of progesterone (P4) and its preferred substrate 17α-hydroxyprogestrone (17OHP4). CYP21A2 activities, which are required for cortisol and aldosterone biosynthesis, involve the formation of energetically disfavored primary carbon radicals. Therefore, we hypothesized that the binding of P4 and 17OHP4 to CYP21A2 restricts access of the reactive heme-oxygen complex to the C-21 hydrogen atoms, suppressing oxygenation at kinetically more favorable sites such as C-17 and C-16, which are both hydroxylated by cytochrome P450c17 (CYP17A1). We reasoned that expansion of the CYP21A2 substrate-binding pocket would increase substrate mobility and might yield additional hydroxylation activities. We built a computer model of CYP21A2 based principally on the crystal structure of CYP2C5, which also 21-hydroxylates P4. Molecular dynamics simulations indicate that binding of the steroid nucleus perpendicular to the plane of the CYP21A2 heme ring limits access of the heme oxygen to the C-21 hydrogen atoms. Residues L107, L109, V470, I471, and V359 were found to contribute to the CYP21A2 substate-binding pocket. Mutation of V470 and I471 to alanine or glycine preserved P4 21-hydroxylase activity, and mutations of L107 or L109 were inactive. Mutations V359A and V359G, in contrast, acquired 16α-hydroxylase activity, accounting for 40% and 90% of the P4 metabolites, respectively. We conclude that P4 binds to CYP21A2 in a fundamentally different orientation than to CYP17A1 and that expansion of the CYP21A2 substrate-binding pocket allows additional substrate trajectories and metabolic switching.     

Multiplicity of progesterone 21-hydroxylase activities associated with constitutive forms of rabbit liver cytochrome P-450

Constitutive cytochromes P-450 have been solubilized from untreated outbred New Zealand White rabbit liver microsomes. Gradient phosphate buffer elution of DEAE-cellulose columns partially resolved six P-450 fractions. Progesterone 21-hydroxylase activity was reconstituted with several fractions and inhibited by an antibody towards P-450 Form 1. One fraction (LM3b) preferentially catalysed the 6 beta- and 16 alpha-hydroxylation of progesterone. SDS-PAGE indicated the presence of proteins with mobilities closely related to Form 1 in several fractions that were separated from this isozyme by DEAE-cellulose chromatography. These results suggest that several constitutive P-450 fractions may contribute to the regiospecific 21-hydroxylation of progesterone.     

Nucleotide sequence of the unique nitrate/nitrite-inducible cytochrome P-450 cDNA from Fusarium oxysporum

A cDNA clone for the nitrate/nitrite-inducible cytochrome P-450 (P-450) of the fungus Fusarium oxysporum (tentatively termed P-450dNIR) was isolated by an immunoscreening method. Sequence determination revealed a polypeptide of 403 amimo acid residues (Mr = 44,371), which was shown to contain the full-length sequence of the fungal P-450. The amino terminus region of the predicted sequence contained neither the signal-like, hydrophobic domain that is commonly observed in microsomal P-450s nor the tagging prosequence that is essential for localization of mitochondrial P-450s. Further, the sequence exhibited higher homologies against those of soluble bacterial P-450s, in particular P-450s of Streptomyces, rather than those of eukaryotic P-450s including yeast and fungal P-450s. These results are highly indicative that P-450dNIR is the first soluble P-450 derived from eukaryotic organisms. The unique features might be related to the novel function of P-450dNIR, which is involved in a dissimilatory reduction of nitrite by the fungus. P-450dNIR was classified into a new family, P-450LV, and the corresponding gene of the fungus was named CYP55.     

Olfactory-specific cytochrome P-450. cDNA cloning of a novel neuroepithelial enzyme possibly involved in chemoreception

We isolated cDNA clones for cytochrome P-450 genes expressed in the olfactory neuroepithelium by screening a corresponding rat cDNA library. Sequence analysis and RNA blot hybridization revealed a new cytochrome P-450, designated cytochrome P-450olf1, which is the first reported cytochrome P-450 mRNA uniquely expressed in the chemosensory organ. Cytochrome P-450olf1 shows intermediate level of sequence similarity (38-53% identity) to several liver cytochrome P-450 enzymes, suggesting that it belongs to the cytochrome P-450II family, but defines a new subfamily (cytochrome P-450IIG) within it. Cytochrome P-450II enzymes are known to process diverse organic compounds, including odorants. This, together with the specificity of cytochrome P-450olf1 to the sensory neuroepithelium, may indicate a role for this protein in olfactory reception.     

Identification of rabbit microsomal cytochrome P-450 isozyme,form 1, as a hepatic progesterone 21-hydroxylase

Six highly purified forms of rabbit microsomal cytochrome P-450, isolated from hepatic microsomes, exhibit differences in the regiospecific metabolism of progesterone. Only one of the isozymes studied, form 1, catalyzes the formation of deoxycorticosterone from progesterone at an appreciable rate. This cytochrome P-450 isozyme may participate in the conversion of progesterone to deoxycorticosterone during pregnancy. All six forms of cytochrome P-450 catalyze 6β- and 16α-hydroxylation at the two concentrations of progesterone tested. Form 3b exhibits a lower apparent Km for 6β-hydroxylation than the other five.     

Nucleotide sequence of a full-length cDNA coding for 3-methylcholanthrene-induced rat liver cytochrome P-450MC

We constructed a full-length cDNA coding for 3-methylcholanthrene-inducible rat liver cytochrome P-450MC by the method of Okayama and Berg. The isolated clone pAU157 contained the cDNA insert of 2.7 kb in length. Sequence analysis of the cDNA insert revealed that the amino acid sequence of cytochrome P-450MC was composed of 523 amino acid residues, including the initial 22 N-terminal amino acids whose sequence was determined with the purified protein. The primary structure was found to contain two highly conserved regions as pointed out from comparisons of the reported amino acid sequences of cytochrome P-450 species. The predicted molecular weight of the apoprotein was 59,300 daltons. Therefore, we concluded that the amino acid sequence determined here is for cytochrome P-450MC, probably corresponding to cytochrome P-450c.     

Effect of spin state on reduction of cytochrome P-450 (P-450C21) from bovine adrenocortical microsomes

The effect of spin state on cytochrome P-450 reduction was studied with a reconstituted system consisting of P-450C21 and NADPH-cytochrome P-450 reductase (NADPH:ferricytochrome oxidoreductase, EC 1.6.2.4) purified from bovine adrenocortical microsomes. The absolute high spin contents of substrate-free, progesterone-bound and 17 alpha-hydroxyprogesterone-bound P-450C21 were estimated from the analysis of thermally induced difference spectra to be 25, 78 and 94% at 25 degrees C, respectively, in 50 mM potassium phosphate buffer (pH 7.2) containing 20% glycerol, 0.1 mM EDTA and 0.5% Emulgen 913. The effect of the high spin content on P-450C21 reduction by NADPH in the reconstituted system was analyzed by a steady-state method and by a stopped-flow method at 25 degrees C. The steady-state results showed that the rate of P-450C21 reduction was not affected by the high spin content of substrate-bound P-450C21 but was very slow without a steroid substrate. Biphasic reduction of P450C21 containing two first-order processes was observed in the stopped-flow experiment in the presence of either of the steroid substrates, but the reduction was very slow without the substrate. There were no significant differences in the rate and the amount of the fast phase of reduction between 17 alpha-hydroxyprogesterone-bound and progesterone-bound P-450C21. Both kinetic studies indicate that the spin state does not control the electron transfer from NADPH to P-450C21 via NADPH-cytochrome P-450 reductase but the presence of substrate is essential for the reduction of P-450C21.     

Immunolocalization of cholesterol side-chain-cleavage cytochrome P-450 and 17 alpha-hydroxylase cytochrome P-450 in bovine ovarian follicles

Follicles were collected from cows and processed for electron microscopy and for immunofluorescent staining at the light microscope level. Key regulatory steroidogenic enzymes cholesterol side-chain-cleavage cytochrome P-450 (P-450scc) and 17 alpha-hydroxylase cytochrome P-450 (P-45017 alpha) were immunolocalized using specific IgG fractions raised against these enzymes. In larger follicles in which the theca interna had differentiated, positive staining for cytochromes P-450scc and P-450(17) alpha was observed in the cells of the theca interna. Electron microscopic examination showed that these cells were rich in endoplasmic reticulum, mainly rough, and had moderate numbers of mitochondria with tubular and lamellar cristae. Positive staining was also present in the theca of follicles undergoing atresia. Positive staining for cytochrome P-450(17) alpha was not observed in the membrana granulosa but cytochrome P-450scc was present in the membrana granulosa in some follicles, particularly in the larger antral follicles. By contrast, positive staining for both enzymes was not observed in stroma, surface epithelium or in small preantral follicles in which the theca interna had not differentiated. These results indicate good agreement between the type(s) of steroidogenic enzyme(s) present in tissues and the type(s) of steroid hormone(s) produced. It is concluded that regulation of steroid hormone production involves, at least in part, regulation of the levels of steroidogenic enzymes.     

Cloning and sequence determination of a complementary DNA related to human liver microsomal cytochrome P-450 S-mephenytoin 4-hydroxylase

A cDNA sequence related to the human cytochrome P-450 responsible for S-mephenytoin 4-hydroxylation (P-450MP) has been isolated from a human liver bacteriophage lambda gt11 library with antibodies specific for P-450MP. The total length of the cDNA is 2.5 kilobases (kb), of which there is a 1.6-kb EcoRI fragment coding for all but five amino acids corresponding to the N-terminus of the protein and including a small noncoding region at the 3' end. This 1.6-kb fragment has been sequenced and used as a probe to analyze human genomic DNA and liver RNA. The sequence shows extensive sequence similarity with that of rabbit liver cytochrome P-450 progesterone 21-hydroxylase [Tukey, R. H., Okino, S., Barnes, H., Griffin, K. J., & Johnson, E. F. (1985) J. Biol. Chem. 260, 13347-13354], and this cDNA, like the rabbit clone, appears to be part of a multigene family. At least two liver mRNA species, 2.2 kb and 3.5 kb, hybridize to the cDNA sequence. The cloning of this gene should aid in analyzing the molecular basis for the genetic polymorphism of S-mephenytoin 4-hydroxylation reported in humans.     

Complete nucleotide sequence of a methylcholanthrene-inducible cytochrome P-450 (P-450d) gene in the rat

The rat cytochrome P-450d gene which is inducibly expressed by the administration of 3-methylcholanthrene (MC) has been cloned and analyzed for the complete nucleotide sequence. The gene is 6.9 kilobases long and is separated into 7 exons by 6 introns. The insertion sites of the introns in this gene are well-conserved as compared with those of another MC-inducible cytochrome P-450c gene, but are completely different from those of a phenobarbital-inducible cytochrome P-450e gene. The overall homologies in the coding nucleotide and deduced amino acid sequences were 75% and 68% between the two MC-inducible cytochrome P-450 genes, respectively. The similarity of the gene organization between cytochrome P-450d and P-450c as well as their homology in the deduced amino acid and the nucleotide sequences suggests that these two genes of MC-inducible cytochromes P-450 constitute a different subfamily than those of the phenobarbital-inducible one in the cytochrome P-450 gene family. In contrast with the notable sequence homology in the coding region of the two MC-inducible cytochromes P-450, all the introns and the 5'- and 3'-flanking regions of the two genes showed virtually no sequence homology between them except for several short DNA segments that are located in the promoter region and the first intron. The nucleotide sequences and the locations of these conserved short DNA segments in the two genes suggest that they may affect the expression of the genes. Middle repetitive sequence reported as ID or identifier sequence were found in and in the vicinity of the cytochrome P-450d gene.     

Cloned cytochrome P-450 cDNA. Nucleotide sequence and homology to multiple phenobarbital-induced mRNA species

Phenobarbital (PB) treatment of rats of various strains leads to the accumulation of liver mRNAs which encode two or three immunochemically related but electrophoretically separable cytochrome P-450 polypeptides. These mRNAs hybridize efficiently to a single cloned cDNA derived from mRNA of PB-treated rats and, therefore, must have extensive sequence homology. The nucleotide sequence of this cloned cDNA was determined and shown to encode the COOH-terminal 211 amino acids of one of the major cytochrome P-450 isozymes induced in rat liver by PB. Together with the recently reported sequence data of Fujii-Kuriyama et al. (Fujii-Kuriyama, Y., Mizukami, Y., Kawajiri, K., Sagawa, K., and Muramatsu, M. (1982) Proc. Natl. Acad. Sci. U. S. A. 79, 2793-2797) for cloned rat cytochrome P-450 cDNA, our data suggest that differences between two closely related P-450 isozymes are restricted to the COOH-terminal half of the polypeptides, with highly divergent regions flanking a tridecapeptide which has been previously shown to be highly conserved in two dissimilar forms of rabbit liver cytochrome P-450. The significance of other interesting features of the cDNA sequence such as a second long (409 residues) open frame, an unusual poly(A) addition signal, and the absence of long hydrophobic stretches in the encoded polypeptide is discussed.     

Purification of cytochrome P-450 from bovine adrenocortical mitochondria

One soluble cytochrome P.450 from bovine adrenocortical mitochondria has been purified to near homogeneity. The purified enzyme catalyses side-chain cleavage of cholesterol and to a much lesser extent 11β-hydroxylation (<13% side-chain cleavage) but shows no 18-hydroxylase activity. The molecular weight of this P.450 is approximately 800,000.     

Structural analysis of cloned cDNA for mRNA of microsomal cytochrome P-450(C21) which catalyzes steroid 21-hydroxylation in bovine adrenal cortex

We have isolated cDNA clones of the mRNA for cytochrome P-450 that catalyzes the steroid C-21 hydroxylation (P-450(C21)), which specifically catalyzes 21-hydroxylation of steroids in the microsomes of bovine adrenal cortex by using synthetic oligonucleotides as probes. Sequence determination of the cloned cDNA showed that it contains 2157 nucleotides and a poly(A) chain and that a single open reading frame of 1488 nucleotides codes for a polypeptide of 496 amino acids with a molecular weight of 56,113. The deduced amino acid composition is in agreement with that determined by direct amino acid analysis of purified P-450(C21) and the predicted primary structure contained amino acid sequences of N-terminal region and two internal tryptic fragments of the protein so far analyzed. Comparing the amino acid sequence with those of other forms of P-450 reveals that a conserved amino acid sequence containing a putative heme-binding cysteine is present in the equivalent position, proximate to the COOH terminus of the molecules and that P-450(C21) is phylogenically situated in an intermediate position between steroidogenic mitochondrial cytochrome P-450 which catalyzes the side-chain cleavage of cholesterol (P-450(SCC)) and drug-metabolizing microsomal P-450s. However, the amino acid sequence of P-450(C21) is much closer to that of drug-metabolizing P-450s than to that of P-450(SCC).