Deformylation of 32-oxo-24,25-dihydrolanosterol by the purified cytochrome P-45014DM (lanosterol 14 alpha-demethylase) from yeast evidence confirming the intermediate step of lanosterol 14 alpha-demethylation

32-Oxo-24,25-dihydrolanosterol (32-oxo-DHL) was deformylated to 4,4-dimethylcholesta-8,14-dien-3 beta-ol, the product of 14 alpha-demethylation of 24,25-dihydro-lanosterol (DHL), by the reconstituted lanosterol 14 alpha-demethylase system consisting of cytochrome P-45014DM and NADPH-cytochrome P-450 reductase of yeast. Affinity of 32-oxo-DHL to the cytochrome was considerably higher than those of lanosterol and DHL, and the rate of deformylation of 32-oxo-DHL was faster than the rate of demethylation of lanosterol and DHL. Spectral analysis of the 32-oxo-DHL complex of cytochrome P-45014DM suggested the interaction between the 32-aldehyde group and the heme iron. These observations, together with our preceding findings on the metabolism of 32-hydroxy-24,25-dihydrolanosterol (Aoyama, Y., Yoshida, Y., Sonoda, Y., and Sato, Y. (1987) J. Biol. Chem. 262, 1239-1243), indicate that the 14 alpha-demethylation of lanosterol catalyzed by cytochrome P-45014DM proceeds with three step monooxygenations via the 32-hydroxy and 32-oxo intermediates, and the cytochrome mediates this sequential reaction without releasing the intermediates.     

Metabolism of 32-hydroxy-24,25-dihydrolanosterol by purified cytochrome P-45014DM from yeast. Evidence for contribution of the cytochrome to whole process of lanosterol 14 alpha-demethylation

Metabolism of 32-hydroxy-24,25-dihydrolanosterol (lanost-8-ene-3 beta,32-diol), a posturated intermediate of the 14 alpha-demethylation (removal of C-32) of 24,25-dihydrolanosterol (lanost-8-en-3 beta-ol), by a reconstituted system consisting of yeast cytochrome P-450 which catalyzes lanosterol 14 alpha-demethylation (cytochrome P-45014DM) (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660 and Aoyama, Y., Yoshida, Y., and Sato, R. (1984) J. Biol. Chem. 259, 1661-1666) and NADPH-cytochrome P-450 reductase was studied. The reconstituted system converted both 32-hydroxy-24,25-dihydrolanosterol and 24,25-dihydrolanosterol to 4,4-dimethyl-5 alpha-cholesta-8,14-dien-3 beta-ol, the 14 alpha-demethylated product of the latter. The metabolism of these compounds was inhibited by a low concentration of ketoconazole which is a potent cytochrome P-45014DM inhibitor. Affinity of cytochrome P-45014DM for 32-hydroxy-24,25-dihydrolanosterol was about 20 times higher than for 24,25-dihydrolanosterol and the cytochrome metabolized the former about 4 times faster than the latter under the experimental conditions. Spectral analysis suggested that the 32-hydroxyl group of 32-hydroxy-24,25-dihydrolanosterol interacted with the heme iron of the oxidized cytochrome and this interaction might support the high affinity of this compound for the cytochrome. These lines of evidence indicate that 32-hydroxy-24,25-dihydrolanosterol is the intermediate of the 14 alpha-demethylation of 24,25-dihydrolanosterol by cytochrome P-45014DM. It is also clear that the cytochrome catalyzes further metabolism of the 32-hydroxylated intermediate to the 14 alpha-demethylated product with higher efficiency than the 32-hydroxylation of the substrate. Cytochrome P-45014DM is thus classified as lanosterol C14-C32 lyase.     

Purification and characterization of cytochrome P-45014DM (lanosterol 14 alpha-demethylase) from pig liver microsomes.

Cytochrome P-45014DM, which catalyzes lanosterol 14 alpha-demethylation, from pig liver microsomes was purified to a state of virtually homogeneous by gel electrophoresis. Its apparent monomeric molecular weight was estimated to be 53,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and the amino-terminal amino acid sequence was Gly-Leu-Leu-Thr-Gly(Leu)-Asp-Leu-Leu-Gly-Ile. When reconstituted with NADPH-cytochrome P-450-reductase, the enzyme showed a high activity for lanosterol and 24,25-dihydrolanosterol 14 alpha-demethylation. Furthermore, the oxygenated intermediates of 24,25-dihydrolanosterol 14 alpha-demethylation, 32-hydroxy-24,25-dihydrolanosterol and 32-oxo-24,25-dihydrolanosterol, were converted to the 32-nor compound, 4,4-dimethylcholesta-8,14-dien-3 beta-ol, by the reconstituted enzyme system.     

Different substrate specificities of lanosterol 14a-demethylase (P-45014DM) of Saccharomyces cerevisiae and rat liver for 24-methylene-24,25-dihydrolanosterol and 24,25-dihydrolanosterol.

The purified lanosterol 14a-demethylase (P-45014DM) of S. cerevisiae catalyzed the 14a-demethylation of 24-methylene-24,25-dihydrolanosterol (24-methylenelanost-8-en-3 beta-ol, 24-methylene-DHL), the natural substrate of the demethylase of filamentous fungi, as well as its natural substrate, lanosterol. Lanosterol 14a-demethylase of rat liver microsomes also catalyzed the 14a-demethylation of 24-methylene-DHL, but the activity was considerably lower than that for lanosterol. The activity of the rat liver enzyme for 24-methylene-DHL was also lower than that for 24,25-dihydrolanosterol (DHL), while the activity of yeast P-45014DM for 24-methylene-DHL was considerably higher than that for DHL. Since 24-substituted sterols are not found in mammals and DHL is not an intermediate of ergosterol biosynthesis by yeast, above-mentioned different substrate specificities between the yeast and the mammalian 14a-demethylases may reflect certain evolutional alteration in their active sites in relation to the difference in their sterol biosynthetic pathways.     

Structural analysis of the interaction between the side-chain of substrates and the active site of lanosterol 14 alpha-demethylase (P-450(14)DM) of yeast.

The role of the side-chain of lanosterol in the enzyme-substrate interaction of yeast P-450(14)DM (lanosterol 14 alpha-demethylase) was analyzed with lanosterol derivatives having functional groups on the side-chain. Purified P-450(14)DM from Saccharomyces cerevisiae catalyzed 14 alpha-demethylation of 26-hydroxylanosterol and 25-hydroxy-24,25-dihydrolanosterol with a lower activity than lanosterol and 24,25-dihydrolanosterol. This enzyme demethylated the (Z)-24-ethylidene-24,25-dihydrolanosterol with a low rate, but did not metabolize the E-isomer. The apparent Km of 26-hydroxylanosterol was 10.8 microM, which was higher than that of lanosterol, but lower than that of 24,25-dihydrolanosterol. On the other hand, competition experiments suggested that the affinity of 25-hydroxy-24,25-dihydrolanosterol and (Z)-24-ethylidene-24,25-dihydrolanosterol for P-450(14)DM was significantly lower than that of 24,25-dihydrolanosterol. Integration of the present results with the preceding ones (Aoyama, Y., Yoshida, Y., Sonoda, Y. and Sato, Y. (1991) Biochim. Biophys. Acta, 1081, 262-266 and Aoyama, Y. and Yoshida, Y. (1991) Biochem. Biophys. Res. Commun., 178, 1064-1071) suggests that yeast P-450(14)DM recognizes two parts of the side-chain, the structure around C-24 and the terminal fork consisting of C-25, C-26 and C-27.     

Induction and substrate specificity of the lanosterol 14 alpha-demethylase from Saccharomyces cerevisiae Y222.

The potential inducibility of the lanosterol 14 alpha-demethylase (P-45014DM) from Saccharomyces cerevisiae Y222 by xenobiotics was investigated. This enzyme and NADPH-cytochrome P-450 reductase were unaffected by a number of compounds known to induce mammalian and some yeast cytochrome P-450 monooxygenases. Furthermore, dibutyryl cyclic AMP did not affect P-45014DM or P-450 reductase levels, while growth at 37 degrees C resulted in a slight decrease. P-45014DM was found to be specific for lanosterol and did not metabolize a number of P-450 substrates including benzo[a]pyrene.     

Role of the side chain of lanosterol in substrate recognition and catalytic activity of lanosterol 14 alpha-demethylase (cytochrome P-450 (14DM)) of yeast

The 14 alpha-demethylation of 24,25-dihydrolanosterol (DHL) derivatives having trimmed side chains, 27-nor-DHL, 26,27-dinor-DHL, 25,26,27-trinor-DHL, 24,25,26,27-tetranor-DHL, 23,24,25,26,27-pentanor-DHL and 22,23,24,25,26,27-hexanor-DHL, was studied with the reconstituted lanosterol 14 alpha-demethylase system consisting of cytochrome P-450(14DM) and NADPH-cytochrome P-450 reductase both purified from yeast microsomes. The demethylase catalyzed the 14 alpha-demethylation of the derivatives having the side chains longer than tetranor but the activities for the trinor- and tetranor-derivatives were lower. Kinetic analysis indicated that affinity of the trinor-derivative for the demethylase was considerably higher than that of DHL. The affinities of the 27-nor- and dinor-derivatives were increased by this order and were the intermediates of DHL and the trinor derivative. On the other hand, Vmax values of the demethylase for the DHL derivatives were decreased depending on their side-chain lengths, and the substrate-dependent reduction rate of cytochrome P-450(14DM) was also decreased in the same manner. Based on these observations, it was concluded that interaction of the side chain of lanosterol especially C-25, 26 and 27 with the substrate site of lanosterol 14 alpha-demethylase was necessary for enhancing the catalytic activity of the enzyme. However, this interaction was considered not to be essential for substrate binding.     

Lanosterol 14 alpha-demethylase (P45014DM): effects of P45014DM inhibitors on sterol biosynthesis downstream of lanosterol

Lanosterol 14 alpha-demethylase (P45014DM) is the cytochrome P450 enzyme complex responsible for an early step in cholesterol biosynthesis, namely the 14 alpha-demethylation of lanosterol. We have synthesized a novel series of steroidal substrate analogues, designed to be specific and potent inhibitors of P45014DM. We describe here the effects of these compounds on sterol biosynthesis downstream from lanosterol, focusing ultimately on their efficacy as inhibitors of cholesterol biosynthesis. Results using a radio-high performance liquid chromatography (HPLC) assay show that in rat liver microsomal preparations, with [24,25-3H]dihydrolanosterol as substrate, the compounds do indeed inhibit the biosynthesis of sterols downstream from lanosterol. A range of inhibitory potencies was observed, and the key enzyme being inhibited was believed to be P45014DM. Inhibitor efficacy was readily correlated with non-metabolized [24,25-3H]dihydrolanosterol, formation of 4,4-dimethyl-cholest-8-en-3 beta-ol, and formation of lathosterol, a sterol believed to be an excellent indicator of whole body cholesterol biosynthesis in humans.     

Spectral properties of a novel cytochrome P-450 of a Saccharomyces cerevisiae mutant SG1. A cytochrome P-450 species having a nitrogenous ligand trans to thiolate

An altered cytochrome P-450 (SG1 P-450) was partially purified from Saccharomyces cerevisiae mutant SG1 which is defective in lanosterol 14 alpha-demethylation. Oxidized SG1 P-450 showed a Soret peak at 422 nm and the alpha peak was lower than the beta peak. This spectrum was considerably different from those of known low-spin P-450s, indicating a unique ligand structure of SG1 P-450. The absorption spectrum of ferric SG1 P-450 was superimposable on that of the imidazole complex of ferric P-450, suggesting the presence of a nitrogenous ligand such as histidine of the apoprotein at the 6th coordination position. SG1 P-450 was immunochemically indistinguishable from cytochrome P-450 of S. cerevisiae catalyzing lanosterol 14 alpha-demethylation (P-45014DM) but had no lanosterol 14 alpha-demethylase activity.     

Evidence for the contribution of a sterol 14-reductase to the 14 alpha-demethylation of lanosterol by yeast

Lanosterol was converted to a 14-demethylated metabolite, 4,4-dimethylzymosterol by Saccharomyces cerevisiae microsomes. This metabolism was mediated by a cytochrome P-450 (P-450/14DM). However, a reconstituted system consisting of P-450/14DM and its reductase converted lanosterol to the 14-desaturated derivative of 4,4-dimethylzymosterol, 4,4-dimethyl-5 alpha-cholesta-8, 14,24-trien-3 beta-ol (trienol). When AY-9944 was added to the reaction system with the microsomes, the trienol was formed with corresponding decrease in 4,4-dimethylzymosterol. These observations indicate that the 14 alpha-demethylation of lanosterol by yeast microsomes occurs sequentially via the trienol. Reduction of the trienol to 4,4-dimethylzymosterol is mediated by an AY-9944-sensitive reductase.     

Yeast cytochrome P-450 catalyzing lanosterol 14 alpha-demethylation. II. Lanosterol metabolism by purified P-450(14)DM and by intact microsomes

A reconstituted monooxygenase system containing a form of cytochrome P-450, termed P-450(14)DM, and NADPH-cytochrome P-450 reductase, both purified from yeast microsomes, catalyzed the conversion of lanosterol (4,4,14 alpha-trimethyl-5 alpha-cholesta-8,24-dien-3 beta-01) to a sterol metabolite in the presence of NADPH and molecular oxygen. This conversion did not occur anaerobically or when either P-450(14)DM, the reductase, or NADPH was omitted from the system. In both free and trimethylsilylated forms, this metabolite showed a relative retention time (relative to lanosterol) of 1.10 in gas chromatography on OV-17 columns. Comparison of its mass spectrum and retention time with those of lanosterol and 4,4-dimethylzymosterol (4,4-dimethyl-5 alpha-cholesta-8,24-dien-3 beta-ol) indicated that the metabolite was 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol. Upon aerobic incubation of microsomes from semianaerobically grown yeast cells in the presence of NADPH and cyanide, endogenous lanosterol was converted to 4,4-dimethylzymosterol. This metabolism was inhibited by CO, metyrapone, SKF-525A, and antibodies to P-450(14)DM. It is concluded that in yeast microsomes lanosterol is 14 alpha-demethylated by a P-450(14)DM-containing monooxygenase system to give rise to 4,4-dimethyl-5 alpha-cholesta-8,14,24-trien-3 beta-ol, which is then reduced to 4,4-dimethylzymosterol by an NADPH-linked reductase.     

Stereoselective interaction of an azole antifungal agent with its target, lanosterol 14 alpha-demethylase (cytochrome P-45014DM): a model study with stereoisomers of triadimenol and purified cytochrome P-45014DM from yeast

The effect of the four triadimenol stereoisomers on the purified yeast lanosterol 14 alpha-demethylase (cytochrome P-45014DM), the primary target of azole antifungal agents, was studied. (1S,2R)-Triadimenol was the most potent demethylase inhibitor and bound quantitatively to the enzyme below 0.05 microM. This isomer also interfered with the chemical reduction of cytochrome P-45014DM and the binding of CO to the cytochrome. The other isomers showed a lower inhibitory effect on the enzyme, and the order of activity was (1R,2R) greater than (1R,2S) greater than or equal to (1S,2S). Based on these findings and the reported preferred conformations for the triadimenol stereoisomers (Anderson, N.H. et al., Pestic. Sci. 15:310-316, 1984), it is predicted that orientation of the hydrophobic tert-butyl and p-chlorophenyl groups relative to the azole nitrogen is important to fit the antifungal agent in the active site of the demethylase.     

Buthiobate: a potent inhibitor for yeast cytochrome P-450 catalyzing 14 alpha-demethylation of lanosterol

Buthiobate (S-n-butyl S'-p-tert-butylbenzyl N-3-pyridyldithiocarbon-imidate), a fungicide, inhibited 14 alpha-demethylation of lanosterol catalyzed by a reconstituted enzyme system consisting of cytochrome P-450 (P-450(14)-DM) and NADPH-cytochrome P-450 reductase both purified from Saccharomyces cerevisiae. Concentration of buthiobate necessary for the 50% inhibition was 0.3 microM and this value was markedly lower than those of metyrapone and SKF-525A. Buthiobate bound stoichiometrically to P-450(14)-DM and induced Type II spectral change of the cytochrome. Buthiobate inhibited lanosterol-dependent enzymatic reduction of the cytochrome. These facts indicate that buthiobate binds to P-450(14)-DM with high affinity and acts as a potent inhibitor on the cytochrome.     

Isolation and characterization of an altered cytochrome P-450 from a yeast mutant defective in lanosterol 14 alpha-demethylation

A cytochrome P-450 (P-450SG1) was purified from a lanosterol 14 alpha-demethylase (P-450(14DM)) defective mutant of Saccharomyces cerevisiae, strain SG1, by a method similar to that used in the purification of the wild type enzyme (Yoshida, Y., and Aoyama, Y. (1984) J. Biol. Chem. 259, 1655-1660). P-450SG1 had the same apparent Mr as and was immunochemically identical to P-450(14DM). Peptide maps of P-450SG1 made by limited proteolysis with Staphylococcus aureus V8 proteinase, chymotrypsin, or papain followed by gel electrophoresis were identical to corresponding peptide maps of P-450(14DM). However, P-450SG1 showed no lanosterol 14 alpha-demethylase activity and its mode of interaction with diniconazole [(E)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-1-y1)-1- penten-3- o1], a specific inhibitor of P-450(14DM), was fundamentally different from that of P-450(14DM). The absorption spectrum of ferric P-450SG1 was unusual for a native low-spin cytochrome P-450 and was superimposable on that of 1-methylimidazole complex of P-450(14DM), indicating that P-450SG1 has a histidine 6th ligand trans to the thiolate 5th ligand, while the 6th ligand of other ferric low-spin cytochrome P-450s is a water molecule or a hydroxyl group of an oxyamino acid. It is concluded that P-450SG1 is an altered P-450(14DM). Difference in the primary structure between P-450SG1 and P-450(14DM) may be slight and was not detected by peptide mapping. However, the alteration caused significant change in the substrate site and heme environments of the cytochrome. P-450SG1 is the first example of a cytochrome P-450 having a histidine axial ligand trans to thiolate and of a genetically altered cytochrome P-450 isolated in a homogeneous state.     

Transport of electrons from mitochondria to microsomes in the reconstituted system of cell organelles

The reduction of cytochrome P-450--CO complex in the presence of various agents in the reconstituted system of liver cell organelles was studied. The reconstituted system was obtained by the preincubation of isolated liver microsomes and mitochondria of the rats kept on a prolonged phenobarbital diet. The addition of glutamate (but not succinate), NAD+ and amytal (or rotenone) to the reconstituted system caused a 40-50% reduction of NADPH-reducible cytochrome P-450. The inhibitor of mitochondrial NADH-cytochrome b5 reductase dicumarol prevented the cytochrome P-450 reduction in the presence of glutamate, NAD+ and amytal but did not affect the reduction of cytochrome P-450 by the added NADH. It was concluded that the electron transfer from the NAD-dependent substrates of the inner mitochondrial respiratory chain to the microsomal cytochrome P-450 occurs with the participation of non-bound NAD and cytochrome b5 of the outer mitochondrial membrane on the condition that the membranes of the two main oxidative systems are in tight contact.     

Cytochrome P-450-dependent oxidation of lanosterol in cholesterol biosynthesis. Microsomal electron transport and C-32 demethylation

Electron transfer to rat liver microsomal cytochrome P-450 of 14 alpha-methyl group demethylation of 24,25-dihydrolanosterol (C30-sterol) has been studied with a new radio-high-performance liquid chromatography assay. The monooxygenase is dependent upon NADPH plus oxygen, insensitive to CN-, and sensitive to CO. Microsomal oxidation is also sensitive to trypsin digestion, and reactivation is dependent upon the addition of purified, detergent-solubilized cytochrome P-450 reductase. Electron transport of C-32 sterol demethylation can be fully supported by very low concentrations of NADPH (approximately 10 microM) only in the presence of saturating concentrations of NADH (approximately 200 microM) suggesting involvement of cytochrome b5-dependent electron transfer in addition to the NADPH-supported pathway. The cytochrome P-450 of 14 alpha-demethylation has been solubilized with detergents, resolved chromatographically from cytochrome P-450 reductase and cytochrome b5, and fully active C-32 demethylase reconstituted. Incubation of intact microsomes with NADH and very low concentrations of NADPH described above leads to interruption of demethylation without 14 alpha-methyl group elimination. Under these conditions, C-32 oxidation products of the C30-sterol substrate accumulate at the expense of formation of demethylated, C29-sterol products. This enzymic interruption of C-32 demethylation, accumulation of oxygenated C30-sterols, along with subsequent demethylation of the isolated C30-oxysterols under similar oxidative conditions supports the suggestion that 14 alpha-hydroxymethyl and aldehydic sterols are metabolic intermediates of sterol 14 alpha-demethylation. Only very modest inductions of the constitutive cytochrome P-450 isozyme of 14 alpha-methyl sterol oxidase can be obtained with just 2 out of 12 known, potent inducers of mammalian hepatic cytochrome P-450s. Alternatively, administration of complete adjuvant in mineral oil drastically reduces amounts of total microsomal cytochrome P-450 while activity of 14 alpha-methyl sterol oxidase is not affected dramatically. Thus, as much as 2.5-fold enhancement of C-32 oxidase specific activity is obtained when expressed per unit of cytochrome P-450.     

Stereo-selective interaction of enantiomers of diniconazole, a fungicide, with purified P-450/14DM from yeast

R(-) isomer of diniconazole [S-3308L, (E)-1-(2,4-dichlorophenyl)-4,4-dimethyl-2-(1,2,4-triazol-l-yl)-1-+ ++penten-3-ol], a newly developed fungicide strongly inhibited lanosterol 14 alpha-demethylation catalyzed by a yeast cytochrome P-450 (P-450/14DM). On the other hand, S(+) isomer of diniconazole was a weaker inhibitor for P-450/14DM. The R(-) isomer combined with both ferric and ferrous P-450/14DM and interfered binding of CO to the cytochrome. The S(+) isomer also interacted with both forms of P-450/14DM but the absorption spectra of the S(+)-diniconazole complexes were different from those of the R(-)-diniconazole complexes. Furthermore, S(+) isomer did not significantly interfere the binding of CO to P-450/14DM. These observations suggest that P-450/14DM discriminates enantiomers of diniconazole and the R(-) isomer is more favorably fit for the active site of the cytochrome.     

Purification and characterization of rat sterol 14-demethylase P450 (CYP51) expressed in Escherichia coli.

Sterol 14-demethylase P450 (CYP51) is an essential enzyme for sterol biosynthesis by eukaryotes. We have cloned rat and human CYP51 cDNAs [Aoyama, Y., Noshiro, M., Gotoh, O., Imaoka, S., Funae, Y., Kurosawa, N., Horiuchi, T., and Yoshida, Y. (1996) J. Biochem. 119, 926-933]. The cloned rat CYP51 cDNA was expressed in Escherichia coli with modification of the N-terminal amino acid sequence, and the expressed protein (CYP51m) was purified to gel-electrophoretic homogenity. The spectrophotometrically determined specific content of CYP51m was 16 nmol/mg protein and the apparent molecular weight was estimated to be 53,000 on SDS-PAGE. Soret peaks of the oxidized and reduced CO-complex of CYP51m were observed at 417 and 447 nm, respectively. The purified CYP51m catalyzed the 14-demethylation of lanosterol and 24,25-dihydrolanosterol upon reconstitution with NADPH-P450 reductase purified from rat liver microsomes. The apparent K(m) and V(max) values for lanosterol were 10.5 microM and 13.9 nmol/min/nmol P450, respectively, and those for 24, 25-dihydrolanosterol were 20.0 microM and 20.0 nmol/min/nmol P450, respectively. The lanosterol demethylase activity of the reconstituted system of CYP51m was inhibited by ketoconazole, itraconazole and fluconazole with apparent IC(50) values of 0.2, 0.7, and 160 microM, respectively.     

Highly purified microsomal P-450: the oxyferro intermediate stabilized at low temperature.

The oxyferro intermediate of highly purified microsomal P-450 from rabbit liver was formed and stabilized at ?30°C in a mixture of aqueous buffer and glycerol ($\text{1}\text{1}$). Absolute and difference (Fe^2·+O₂-Fe³⁺) spectra of this intermediate appear to be very similar to those obtained under either steady state kinetics or stopped flow conditions on the same cytochrome as well as on bacterial P-450_cam. (Absolute and difference spectra present maxima at 420 and 557–558 nm and a broad maximum at 442 nm respectively). As temperature increases the oxyferro intermediate autoxidizes and ferric cytochrome P-450 is restored. This reaction appears to follow biphasic first order kinetics. The rate constant of both phases decreases with temperature and increases with protons concentrations.     

Mouse knockout of the cholesterogenic cytochrome P450 lanosterol 14alpha-demethylase (Cyp51) resembles Antley-Bixler syndrome

Antley-Bixler syndrome (ABS) represents a group of heterogeneous disorders characterized by skeletal, cardiac, and urogenital abnormalities that have frequently been associated with mutations in fibroblast growth factor receptor 2 or cytochrome P450 reductase genes. In some ABS patients, reduced activity of the cholesterogenic cytochrome P450 CYP51A1, an ortholog of the mouse CYP51, and accumulation of lanosterol and 24,25-dihydrolanosterol has been reported, but the role of CYP51A1 in the ABS etiology has remained obscure. To test whether Cyp51 could be involved in generating an ABS-like phenotype, a mouse knock-out model was developed that exhibited several prenatal ABS-like features leading to lethality at embryonic day 15. Cyp51(-/-) mice had no functional Cyp51 mRNA and no immunodetectable CYP51 protein. The two CYP51 enzyme substrates (lanosterol and 24,25-dihydrolanosterol) were markedly accumulated. Cholesterol precursors downstream of the CYP51 enzymatic step were not detected, indicating that the targeting in this study blocked de novo cholesterol synthesis. This was reflected in the up-regulation of 10 cholesterol synthesis genes, with the exception of 7-dehydrocholesterol reductase. Lethality was ascribed to heart failure due to hypoplasia, ventricle septum, and epicardial and vasculogenesis defects, suggesting that Cyp51 deficiency was involved in heart development and coronary vessel formation. As the most likely downstream molecular mechanisms, alterations were identified in the sonic hedgehog and retinoic acid signaling pathways. Cyp51 knock-out mice provide evidence that Cyp51 is essential for embryogenesis and present a potential animal model for studying ABS syndrome in humans.