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The annotated genome of Streptomyces coelicolor A3(2) revealed 18 cytosolic cytochromes P450 (CYPs) with six ferredoxin (fdx) proteins and two soluble ferredoxin reductases (fpr), their putative electron transport proteins. mRNA expression was observed for all 18 CYPs throughout growth and secondary metabolism, from 3 h after spore germination, and all CYP proteins examined also were present. Expression of members of the fdx complement was detected from the same time point, yet both fpr were detected only at 12 h. Six-hour exposure to dimethylbenzanthracene and benzo[a]pyrene xenobiotics resulted in the absence of some CYP mRNAs and expression of a specific fpr, FR2. This finding and the expression pattern during growth suggested that CYP activity may be regulated by availability of specific reductases. To test this proposal, we expressed in Escherichia coli and purified to homogeneity five CYPs: CYP105D5 (involved in xenobiotic metabolism) and CYP154A1, CYP154C1, CYP158A1, and CYP158A2 (putatively involved in secondary metabolism). Also the two soluble fpr (FR2 and FR3) proposed to shuttle electrons by means of fdx were purified, and specific interactions were observed so that FR2 preferentially reduced CYP105D5 (>90% reduction) compared with the other CYPs (>20% reduction), whereas FR3 preferentially reduced the other CYPs (>85% reduction) compared with CYP105D5 (>10%). Furthermore FR2 was shown to efficiently bind CYP105D5 and drive benzo[a]pyrene hydroxylation in contrast to FR3. These data show that control of CYP activity in S. coelicolor A3(2) involves specific interactions with fpr and their availability during the life cycle and, after xenobiotic exposure, represents a unique mechanism for regulating CYP function.

Hypertension is a leading cause of cardiovascular, cerebral, and renal disease morbidity and mortality. Here we show that disruption of the Cyp 4a14 gene causes hypertension, which is, like most human hypertension, more severe in males. Male Cyp 4a14 (-/-) mice show increases in plasma androgens, kidney Cyp 4a12 expression, and the formation of prohypertensive 20-hydroxyarachidonate. Castration normalizes the blood pressure of Cyp 4a14 (-/-) mice and minimizes Cyp 4a12 expression and arachidonate omega-hydroxylation. Androgen replacement restores hypertensive phenotype, Cyp 4a12 expression, and 20-hydroxy-arachidonate formation. We conclude that the androgen-mediated regulation of Cyp 4a arachidonate monooxygenases is an important component of the renal mechanisms that control systemic blood pressures. These results provide direct evidence for a role of Cyp 4a isoforms in cardiovascular physiology, establish Cyp 4a14 (-/-) mice as a monogenic model for the study of cause/effect relationships between blood pressure, sex hormones, and P450 omega-hydroxylases, and suggest the human CYP 4A homologues as candidate genes for the analysis of the genetic and molecular basis of human hypertension.

Cytochrome P450 14alpha-sterol demethylases (CYP51) are essential enzymes in sterol biosynthesis in eukaryotes. CYP51 removes the 14alpha-methyl group from sterol precursors such as lanosterol, obtusifoliol, dihydrolanosterol, and 24(28)-methylene-24,25-dihydrolanosterol. Inhibitors of CYP51 include triazole antifungal agents fluconazole and itraconazole, drugs used in treatment of topical and systemic mycoses. The 2.1- and 2.2-A crystal structures reported here for 4-phenylimidazole- and fluconazole-bound CYP51 from Mycobacterium tuberculosis (MTCYP51) are the first structures of an authentic P450 drug target. MTCYP51 exhibits the P450 fold with the exception of two striking differences-a bent I helix and an open conformation of BC loop-that define an active site-access channel running along the heme plane perpendicular to the direction observed for the substrate entry in P450BM3. Although a channel analogous to that in P450BM3 is evident also in MTCYP51, it is not open at the surface. The presence of two different channels, with one being open to the surface, suggests the possibility of conformationally regulated substrate-in/product-out openings in CYP51. Mapping mutations identified in Candida albicans azole-resistant isolates indicates that azole resistance in fungi develops in protein regions involved in orchestrating passage of CYP51 through different conformational stages along the catalytic cycle rather than in residues directly contacting fluconazole. These new structures provide a basis for rational design of new, more efficacious antifungal agents as well as insight into the molecular mechanism of P450 catalysis.

Sterol 14alpha-demethylase encoded by CYP51 is a mixed-function oxidase involved in sterol synthesis in eukaryotic organisms. Completion of the Mycobacterium tuberculosis genome project revealed that a protein having homology to mammalian 14alpha-demethylases might be present in this bacterium. Using genomic DNA from mycobacterial strain H(37)Rv, we have established unambiguously that the CYP51-like gene encodes a bacterial sterol 14alpha-demethylase. Expression of the M. tuberculosis CYP51 gene in Escherichia coli yields a P450, which, when purified to homogeneity, has the predicted molecular mass, ca. 50 kDa on SDS/PAGE, and binds both sterol substrates and azole inhibitors of P450 14alpha-demethylases. It catalyzes 14alpha-demethylation of lanosterol, 24, 25-dihydrolanosterol, and obtusifoliol to produce the 8,14-dienes stereoselectively as shown by GC/MS and (1)H NMR analysis. Both flavodoxin and ferredoxin redox systems are able to support this enzymatic activity. Structural requirements of a 14alpha-methyl group and Delta(8(9))-bond were established by comparing binding of pairs of sterol substrate that differed in a single molecular feature, e.g., cycloartenol paired with lanosterol. These substrate requirements are similar to those established for plant and animal P450 14alpha-demethylases. From the combination of results, the interrelationships of substrate functional groups within the active site show that oxidative portions of the sterol biosynthetic pathway are present in prokaryotes.

When the cDNA encoding bovine microsomal 17 alpha-hydroxylase cytochrome P450 (P45017 alpha) containing modifications within the first seven codons which favor expression in Escherichia coli is placed in a highly regulated tac promoter expression plasmid, as much as 16 mg of spectrally detectable P45017 alpha per liter of culture can be synthesized and integrated into E. coli membranes. The known enzymatic activities of bovine P45017 alpha can be reconstituted by addition of purified rat liver NADPH-cytochrome P450 reductase to isolated E. coli membrane fractions containing the recombinant P45017 alpha enzyme. Surprisingly, it is found that E. coli contain an electron-transport system that can substitute for the mammalian microsomal NADPH-cytochrome P450 reductase in supporting both the 17 alpha-hydroxylase and 17,20-lyase activities of P45017 alpha. Thus, not only can E. coli express this eukaryotic membrane protein at relatively high levels, but as evidenced by metabolism of steroids added directly to the cells, the enzyme is catalytically active in vivo. These studies establish E. coli as an efficacious heterologous expression system for structure-function analysis of the cytochrome P450 system.

Ammonium sulfate, as well as potassium phosphate, can be used to measure solubility differences between hemoglobin S and hemoglobin A. In addation, the solubility of deoxyhemoglobin C(Harlem) in 1.96 M phosphate has a markedly different temperature dependence from that of deoxyhemoglobin S. This observation indicates that the solubility measurement is quite sensitive to changes in protein structure. Because of the large degree of comparability between the solubility and the aggregation of deoxyhemoglobin S, solubility was used to measure the effectiveness of organic compounds as noncovalent modifiers of deoxyhemoglobin S aggregation. Organic solvents (ethanol, dimethylsulfoxide, 1,4-dioxane, dimethylformamide) alter the solubility characteristics of deoxyhemoglobin S in 1.96 M phosphate buffer, pH 7.0. The concentrations of solvent necessary to provide a half-maximal effect are remarkably similar (about 0.5 M), although the chemical nature of these compounds is quite different. The effect of these solvents must be to prevent the noncovalent bond formation necessary to produce the insoluble hemoglobin precipitate, perhaps by altering the water structure around the deoxyhemoglobin S molecules. In addition to these organic solvents, guanidine hydrochloride and urea, two well-known protein denaturants, were studied. Guanidine hydrochloride was as effective as the best organic solvent in increasing the solubility of deoxyhemoglobin S; urea was far less effective. Studies in vitro with intact erythrocytes from individuals homozygous for hemoglobin S showed that sickling is decreased up to 50% by treatment with ethanol. This offers further evidence that solubility is monitoring a phenomenon similar to the aggregation of deoxyhemoglobin S inside erythrocytes. While use of these particular compounds in vitro would seem to have no clinical implications, these studies do suggest that the use of chemicals that do not modify hemoglobin S covalently should be explored in efforts to prevent deoxyhemoglobin S aggregation.

Transfected, nonsteroidogenic COS-1 cells derived from monkey kidney are found to be capable of supporting the initial and rate-limiting step common to all steroidogenic pathways, the side-chain cleavage of cholesterol to produce pregnenolone. Endogenous COS-1 kidney cell renodoxin reductase and renodoxin are able to sustain low levels of this activity catalyzed by bovine cholesterol side-chain cleavage cytochrome P450 (P450scc) whose synthesis is directed by a transfected plasmid containing P450scc cDNA. Double transfection with both P450scc and adrenodoxin plasmids leads to greater pregnenolone production and indicates that adrenodoxin plays a role as a substrate for this reaction or that bovine adrenodoxin serves as a better electron donor than the endogenous iron-sulfur protein renodoxin. Also it is found that both the bovine adrenodoxin and P450scc precursor proteins are proteolytically processed upon their uptake by COS-1 cell mitochondria to forms having the same electrophoretic mobility as mature bovine adrenodoxin and P450scc. Following triple transfection of COS-1 cells with P450scc, adrenodoxin, and 17 alpha-hydroxylase cytochrome P450 plasmids, pregnenolone produced in mitochondria by the side-chain cleavage reaction can be further metabolized in the endoplasmic reticulum to 17 alpha-hydroxypregnenolone and dehydroepiandrosterone. Although this functional steroidogenic pathway can be incorporated into this nonsteroidogenic cell type, it is found to be nonresponsive to cAMP, a potent activator of steroid hormone biosynthesis in adrenal cortex, testis, and ovary. Thus the cellular mechanisms necessary to support both microsomal and mitochondrial steroid hydroxylase activities appear not to be tissue specific, whereas the acute cAMP-dependent regulation of steroidogenesis is not present in transformed kidney (COS-1) cells.

Adult bovine adrenal cortical cells in monolayer culture were used to study the induction of cholesterol side-chain-cleavage cytochrome P-450 by corticotropin (ACTH). In the presence of 1 microM ACTH, there was a 4-fold increase in cortisol production by these cells over a 72-hr period and a corresponding increase in total cytochrome P-450 content. The incorporation of [35S]methionine into a number of cellular proteins was stimulated by the presence of ACTH in the culture medium, whereas the incorporation into other proteins was decreased. The temporal profile of these changes varied from one protein to another. Examination of the incorporation of [35S]methionine into mitochondrial protein showed an increased production of a radiolabeled protein that comigrated with the form of cytochrome P-450 known as side-chain-cleavage cytochrome upon incubation with ACTH. Thus, it appears that the cytochrome P-450scc content is increased in bovine adrenal cortical cells exposed to ACTH. Cytochrome P-450scc, synthesized in a cell-free translation system directed by RNA isolated from bovine adrenal cortical tissue or from cells, had a molecular weight of 54,500. Cytochrome P-450scc isolated from bovine adrenal mitochondria had a molecular weight of 49,000. Thus, cytochrome P-450scc is synthesized as a larger precursor that must be processed by proteolytic cleavage before or upon insertion into the mitochondrion.

Two overlapping cDNA clones (pBSCC-1 and pBSCC-2) bearing inserts approximately equal to 425 and approximately equal to 950 base pairs long, respectively, which are specific for bovine cholesterol side-chain cleavage cytochrome P-450 (P-450scc), have been identified by using two differential hybridization screening procedures followed by hybrid-selected RNA translation. By using these cloned cDNAs as hybridization probes, an RNA species was identified that had the properties expected of mRNA specific for P-450scc with respect to tissue specificity, corticotropin (ACTH)-mediated regulation of synthesis, and size of the protein product synthesized in vitro. In RNA samples obtained from bovine adrenal cortex, from bovine corpus luteum, and from cultured bovine adrenocortical cells, it was found that P-450scc is encoded by mRNA species approximately equal to 2000 bases long, a majority of which are polyadenylylated. P-450scc mRNA was not detected in RNA samples prepared from bovine heart, liver, and kidney. Treatment of cultured bovine adrenocortical cells with ACTH resulted in the appearance of elevated levels of P-450scc mRNA within 8 hr. Thus, ACTH promotes the enhancement of P-450scc gene transcription or acts to stabilize the transcripts. When pBSCC-2 cDNA was used to probe high molecular weight bovine DNA following treatment with restriction endonucleases, a simple pattern of hybridization was observed indicating that P-450scc may be encoded by a single gene.

The mechanism of induction of an adult rabbit cytochrome P-450, prostaglandin (PG) omega-hydroxylase (P-450PG-omega), during pregnancy has been investigated. This P-450 isozyme regiospecifically hydroxylates PGE1, PGA1, and PGF2 alpha at carbon-20 (the omega position). The specific activity of this enzyme is induced from 0.07 nmol of 20-OH-PGE1 to 3.05 nmol of 20-OH-PGE1 formed per min per mg of microsomal protein in the lungs of 25- to 28-day pregnant rabbits as compared to nonpregnant rabbits. Immunoblotting studies with a polyclonal antibody raised against this P-450 have shown that there is a concomitant gestational age-dependent increase in the amount of P-450PG-omega microsomal protein accompanying the increase of omega-hydroxylase activities. Within 3 days postpartum, both omega-hydroxylase activity and the amount of immunodetectable P-450PG-omega drop precipitously to near control levels. In vitro translation of total cellular RNA, extracted from the lungs of pregnant rabbits at various days throughout gestation, and immunoprecipitation of newly synthesized P-450PG-omega demonstrated a similar gestational age-dependent increase in translatable P-450PG-omega mRNA, as was observed with omega-hydroxylase activity and P-450PG-omega protein levels. These data suggest that the induction of this P-450 may occur at the transcriptional level and, furthermore, that control of cytochrome P-450PG-omega expression in the rabbit lung is tightly regulated at both protein and mRNA levels.

Several recombinant cDNA clones specific for the mitochondrial iron-sulfur protein adrenodoxin have been identified in a bovine adrenocortical cDNA library. One clone (pBAdx4) contains a 900-base-pair insert that includes the entire amino acid coding region of the adrenodoxin precursor protein. The amino acid sequence of mature adrenodoxin deduced from the nucleotide sequence of pBAdx4 is identical with that determined by protein sequencing except for three amide changes. The previously undetermined sequence of the adrenodoxin NH2-terminal precursor segment (58 amino acids) contains several basic residues, a characteristic feature of the precursor segment of proteins destined for mitochondria. In addition, a 14 amino acid extension is present at the COOH terminus of the mature adrenodoxin sequence. Whether this represents a COOH-terminal precursor segment is not clear. Three different adrenodoxin mRNAs are present [1.75, 1.4, and 0.95 kilobase(s) long] in bovine adrenocortical RNA. RNA from bovine corpus luteum, liver, and kidney contains transcripts that hybridize to adrenodoxin cDNA. Accumulation of adrenodoxin mRNA occurs in cultured bovine adrenocortical cells after treatment with ACTH or dibutyryl-cAMP, similar to that observed for the mitochondrial steroid hydroxylases that it services--namely, the cholesterol side-chain-cleavage cytochrome P-450 and the steroid 11 beta-hydroxylase cytochrome P-450.

Maintenance of optimal steroidogenic capacity in the adrenal cortex is the result of a cAMP-dependent response to the peptide hormone corticotropin (ACTH). The molecular mechanism of this action of ACTH has been examined by using five recombinant DNA clones specific for enzymes of the steroidogenic pathway (P-450scc, P-45011 beta, P-450C21, P-45017 alpha, and adrenodoxin). The presence of nuclear precursors in steady-state RNA samples derived from cultured bovine adrenocortical cells and moderate increases in the number of RNA chain initiations, as determined by in vitro nuclear run-off assays, indicate that ACTH controls the expression of the gene(s) for each of these proteins at the transcriptional level. The ACTH-mediated increase in accumulation of transcripts specific for steroid hydroxylases in nuclear RNA can be specifically blocked by inhibiting protein synthesis in bovine adrenocortical cell cultures. The steady-state concentrations of nuclear RNA for control genes show no decrease upon cycloheximide treatment. These studies suggest that a primary action of ACTH in the adrenal cortex is to activate (via cAMP) the synthesis of rapidly turning over protein factors that in turn mediate increased initiation of transcription of steroid hydroxylase genes. We propose that these protein factors impart specificity of induction to genes encoding components of this pathway in steroidogenic tissues.