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CYP1B1-null mice, created by targeted gene disruption in embryonic stem cells, were born at the expected frequency from heterozygous matings with no observable phenotype, thus establishing that CYP1B1 is not required for mouse development. CYP1B1 was not detectable in cultured embryonic fibroblast (EF) or in different tissues, such as lung, of the CYP1B1-null mouse treated with the aryl hydrocarbon receptor agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin whereas the equivalent wild-type EF cells express basal and substantial inducible CYP1B1 and lung expresses inducible CYP1B1. CYP1A1 is induced to far higher levels than CYP1B1 in liver, kidney, and lung in wild-type mice and is induced to a similar extent in CYP1B1-null mice. 7,12-dimethylbenz[a]anthracene (DMBA) was toxic in wild-type EFs that express CYP1B1 but not CYP1A1. These cells effectively metabolized DMBA, consistent with CYP1B1 involvement in producing the procarcinogenic 3,4-dihydrodiol as a major metabolite, whereas CYP1B1-null EF showed no significant metabolism and were resistant to DMBA-mediated toxicity. When wild-type mice were administered high levels of DMBA intragastrically, 70% developed highly malignant lymphomas whereas only 7.5% of CYP1B1-null mice had lymphomas. Skin hyperplasia and tumors were also more frequent in wild-type mice. These results establish that CYP1B1, located exclusively at extrahepatic sites, mediates the carcinogenicity of DMBA. Surprisingly, CYP1A1, which has a high rate of DMBA metabolism in vitro, is not sufficient for this carcinogenesis, which demonstrates the importance of extrahepatic P450s in determining susceptibility to chemical carcinogens and validates the search for associations between P450 expression and cancer risk in humans.Cytochromes P450 (P450) are a superfamily of hemecontaining monooxygenases. A limited number of P450s participate in pathways of steroid hormone synthesis whereas the majority of these enzymes are involved in oxidative metabolism of drugs, other foreign compounds, and endogenous substrates, including steroids (1). These xenobioticmetabolizing P450s mostly fall within the CYP1, CYP2, and CYP3 families and exhibit broad and sometimes overlapping substrate specificity. A limited number of P450s within these families are responsible for the metabolic activation of chemical carcinogens. In the CYP1 family, CYP1A1 and CYP1B1 metabolically activate polycyclic aromatic hydrocarbons and CYP1A2 participates in the metabolic activation of arylamine, heterocylic amines, and aflatoxin B1. CYP2E1 activates a large number of low molecular weight carcinogens including benzene and N-nitrosodimethylamine. These carcinogenmetabolizing P450s are also among the most well conserved of the P450 superfamily and can be found in several mammalian species, including mouse and human (2). CYP1B1 is a conserved member of the P450 superfamily that was first identified and purified from mouse embryonic fibroblasts (EFs) (3) and rat adrenals (4). This form was characterized by its ability to metabolically act...

Cytochrome P450 (CYP) monooxygenase superfamily contributes a broad array of biological functions in living organisms. In fungi, CYPs play diverse and pivotal roles in versatile metabolism and fungal adaptation to specific ecological niches. In this report, CYPomes in the 47 genomes of fungi belong to the phyla Ascomycota, Basidiomycota, Chytridiomycota, and Zygomycota have been studied. The comparison of fungal CYPomes suggests that generally fungi possess abundant CYPs belonging to a variety of families with the two global families CYP51 and CYP61, indicating individuation of CYPomes during the evolution of fungi. Fungal CYPs show highly conserved characteristic motifs, but very low overall sequence similarities. The characteristic motifs of fungal CYPs are distinguishable from those of CYPs in animals, plants, and especially archaea and bacteria. The four representative motifs contribute to the general function of CYPs. Fungal CYP51s and CYP61s can be used as the models for the substrate recognition sites analysis. The CYP proteins are clustered into 15 clades and the phylogenetic analyses suggest that the wide variety of fungal CYPs has mainly arisen from gene duplication. Two large duplication events might have been associated with the booming of Ascomycota and Basidiomycota. In addition, horizontal gene transfer also contributes to the diversification of fungal CYPs. Finally, a possible evolutionary scenario for fungal CYPs along with fungal divergences is proposed. Our results provide the fundamental information for a better understanding of CYP distribution, structure and function, and new insights into the evolutionary events of fungal CYPs along with the evolution of fungi.

The metabolism of cholesterol by cytochrome P450 side chain cleavage enzyme is hormonally regulated in steroidogenic tissues via intramitochondrial cholesterol transport. The mediating steroidogenic acute regulatory protein (StAR) is synthesized as a 37-kDa (p37) precursor that is phosphorylated by protein kinase A and cleaved within the mitochondria to generate 30-kDa forms (p30, pp30). The effectiveness of modified recombinant StAR forms in COS-1 cells without mitochondrial import has led to a prevailing view that cholesterol transport is mediated by p37 StAR via activity on the outer mitochondrial membrane. The present study of the activation of cholesterol metabolism by bromo-cAMP in adrenal cells in relation to 35 S-StAR turnover indicates that targeting of pp30 to the inner membrane provides the dominant cholesterol transport mechanism. We show that 1) only newly synthesized StAR is functional, 2) phosphorylation and processing of p37 to pp30 occurs rapidly and stoichiometrically, 3) both steps are necessary for optimum transport, and 4) newly synthesized pp30 exhibits very high activity (400 molecules of cholesterol/StAR/min). Segregation of cAMP activation and synthesis of StAR from cholesterol metabolism showed that very low levels of newly synthesized StAR (1 fmol/min/10 6 cells) sustained activated cholesterol metabolism (0.4 pmol/min/10 6 cells, t1 ⁄2 ‫؍‬ 70 min) long after complete removal of p37 (t1 ⁄2 ‫؍‬ 5 min). This activity was highly sensitive to inhibition of processing by CCCP only until sufficient pp30 was formed. Maximum activation preceded bromo-cAMP-induced StAR expression, indicating other limiting steps in cholesterol metabolism.The conversion of cholesterol to pregnenolone, the first step in steroid synthesis, is catalyzed by cytochrome P450 side chain cleavage enzyme (P450scc), 1 which is localized on the matrix side of the inner mitochondrial membrane (1, 2). The transfer of cholesterol to P450scc, a limiting step in this conversion (3, 4), requires hormonal activation of cholesterol mobilization to the mitochondria. This mobilization is dependent on the uptake of lipoproteins, lysosomal-cytosolic transfer, and hydrolysis of cholesterol esters as well as on two subsequent transfer steps (5). The first step involves transfer of cholesterol to the mitochondria and is dependent on an intact cytoskeleton but not on protein synthesis (6). The second step involves transfer within the mitochondria from outer to inner membrane. This trophic hormone-dependent transport of cholesterol from mitochondrial outer membrane to inner membrane (4, 7, 8) is blocked in vivo within 10 min by protein synthesis inhibitors, such as cycloheximide (CHX) (9, 10). A series of cAMP-stimulated and CHX-sensitive phosphoproteins (30 -37 kDa), which localize to the mitochondria, have been identified in cultured adrenal and testis cells (11)(12)(13)(14)(15)(16)(17). The gene that encodes these proteins has been cloned, and the active form has been named the steroidogenic acute regulatory protein (StAR) (18). The exp...