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.     

Multiple regulatory elements control expression of the gene encoding the Saccharomyces cerevisiae cytochrome P450, lanosterol 14 alpha-demethylase (ERG11).

The major cytochrome P450 in the yeast Saccharomyces cerevisiae, lanosterol 14 alpha-demethylase (ERG11), catalyzes an essential reaction in the biosynthesis of ergosterol, the predominant sterol of yeast. Protein levels of this cytochrome P450 are known to be affected by carbon source, oxygen, and heme, as well as the growth state of the culture. We have determined that ERG11 message levels increase during growth on glucose, in the presence of heme, and during oxygen limiting growth conditions and, unexpectedly, during anaerobic growth. To determine the cis-acting regions responsible for regulation of expression of the ERG11 promoter under optimal conditions of fermentative growth, deletion analysis was performed using the Escherichia coli lacZ as a reporter gene. Two upstream activating sequences, UAS1 and UAS2, and an upstream repressor element, URS1, plus a second possible or cryptic repressor element, URS2, were identified in the ERG11 promoter. The HAP1 protein product apparently participates in activation from UAS1 but not from UAS2. Sequences resembling ERG11 UAS2 were identified in seven additional oxygen-regulated genes. Repression of ERG11 expression was dependent upon the ROX1 repressor and additional repressor(s) designated as Old (overexpression of lanosterol demethylase). These data indicate that ERG11 is a member of the hypoxic gene family which includes ANB1, COX5b, CYC7, and HEM13. Furthermore, NADPH-cytochrome P450 reductase (CPR1), another component in this P450 system, appears to be coordinately regulated with ERG11.     

Active site topology of Saccharomyces cerevisiae lanosterol 14 alpha-demethylase (CYP51) and its G310D mutant (cytochrome P-450SG1).

Incubation of phenyldiazene (PhN = NH) with lanosterol 14 alpha-demethylase, a cytochrome P-450 enzyme (CYP51) that oxidatively removes the 14 alpha-methyl group of lanosterol, results in the appearance of a 478-nm band indicative of phenyl-iron complex formation. In situ oxidation of the phenyl-iron complex by ferricyanide yields exclusively the N-phenylprotoporphyrin IX regioisomer with the phenyl group on the nitrogen of pyrrole ring C (NC). The biphenyl-iron complex formed in the analogous reaction of the enzyme with biphenyldiazene similarly rearranges on treatment with ferricyanide to the NC regioisomer of N-biphenylprotoporphyrin IX. The active site cavity must therefore be at least 10 A high directly above the iron atom and pyrrole ring C of the heme group, and lanosterol binds to the enzyme in the region above pyrrole ring C. Phenyl-iron complex formation is not detected spectroscopically with cytochrome P-450SG1, a catalytically inactive G310D mutant of lanosterol 14 alpha-demethylase in which the sixth iron coordination site is thought to be occupied by an imidazole ligand. Nevertheless, oxidation of the phenyldiazene-treated enzyme with ferricyanide provides the NA and NC regioisomers of N-phenylprotoporphyrin IX in a 40:60 ratio. The single amino acid substitution in cytochrome P-450SG1 thus causes a conformational change that retracts the amino acid residues that cover pyrrole ring A and moves an imidazole ligand into the active site.     

Novel cholesterol biosynthesis inhibitors targeting human lanosterol 14alpha-demethylase (CYP51)

Novel cholesterol biosynthesis inhibitors, a group of pyridylethanol(phenylethyl)amine derivatives, were synthesized. Sterol profiling assay in the human hepatoma HepG2 cells revealed that compounds target human lanosterol 14alpha-demethylase (CYP51). Structure-activity relationship study of the binding with the overexpressed human CYP51 indicates that the pyridine binds within the heme binding pocket in an analogy with the azoles.     

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.     

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.     

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.     

Cloning and characterization of the lanosterol 14alpha-demethylase (ERG11) gene in Cryptococcus neoformans

The ergosterol pathway in fungal pathogens is an attractive antimicrobial target because it is unique from the major sterol (cholesterol) producing pathway in humans. Lanosterol 14alpha-demethylase is the target for a major class of antifungals, the azoles. In this study we have isolated the gene for this enzyme from Cryptococcus neoformans. The gene, ERG11, was recovered using degenerate PCR with primers designed with a novel algorithm called CODEHOP. Sequence analysis of Erg11p identified a highly conserved region typical of the cytochrome P450 class of mono-oxygenases. The gene was present in single copy in the genome and mapped to one end of the largest chromosome. Comparison of the protein sequence to a number of major human fungal pathogen Erg11p homologs revealed that the C. neoformans protein was highly conserved, and most closely related to the Erg11p homologs from other basidiomycetes. Functional studies demonstrated that the gene could complement a Saccharomyces cerevisiae erg11 mutant, which confirmed the identity of the C. neoformans gene.     

Regulation of hepatic lanosterol 14 alpha-demethylase gene expression by dietary cholesterol and cholesterol-lowering agents.

Binding of sterol response element binding protein 1a to sterol response element-1 (SRE-1) in the promoter region of lanosterol 14 alpha-demethylase (14DM) has been demonstrated previously. Decreased 14DM activity has been shown to result in accumulation of the intermediate, 3 beta-hydroxy-lanost-8-en-32-al, a known translational downregulator of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase. Since it has also been demonstrated that feedback regulation of hepatic HMG-CoA reductase occurs primarily at the level of translation, the effects of dietary cholesterol and cholesterol lowering agents on levels of hepatic 14DM mRNA and immunoreactive protein were investigated. Addition of 1% cholesterol to a chow diet markedly decreased hepatic 14DM mRNA and protein levels in Sprague-Dawley rats. The extent and time course of this decrease in 14DM immunoreactive protein closely paralleled that of HMG-CoA reductase. Supplementation of the diet with the HMG-CoA reductase inhibitor, Lovastatin, to a level of 0.02%, raised 14DM mRNA and protein levels 2- to 3-fold. Addition of 2% Colestipol, a bile acid binding resin, to the chow diet caused smaller increases. The highest level of 14DM protein expression was observed in liver, the major site of feedback regulation of HMG-CoA reductase by cholesterol. Taken together, these observations suggest a critical role for 14DM in the feedback regulation of hepatic HMG-CoA reductase.     

Isolation of the gene for cytochrome P450L1A1 (lanosterol 14 alpha-demethylase) from Candida albicans

The Saccharomyces cerevisiae cytochrome P450 L1A1 (lanosterol 14 alpha-demethylase)-coding gene was used as a hybridization probe to isolate two HindIII fragments of 2.5 kb and 6.85 kb from a phage lambda library of Candida albicans nucleotide sequences. Restriction endonuclease mapping and Southern blot hybridization experiments indicated that these fragments represent two allelic forms of the same gene. This cloned sequence, when introduced into S. cerevisiae or C. albicans on a multiple copy vector, produced an increase in cytochrome P450 content and resistance to imidazole antifungal agents which are inhibitors of cytochrome P450 L1A1. In addition, the cloned sequence was able to complement a cytochrome P450 L1A1 gene disruption when introduced into S. cerevisiae. These data indicate that the cloned sequence codes for the lanosterol 14 alpha-demethylase cytochrome P450 L1A1 from C. albicans.     

Expression of microsomal lanosterol 14alpha-demethylase (CYP51) in an engineered soluble monomeric form

We prepared a soluble monomeric form of bovine cytochrome P450 lanosterol 14α-demethylase (CYP51), which in mammals is a ubiquitously expressed membrane protein of the endoplasmic reticulum. We constructed two variants of bovine CYP51 (bCYP51) with different truncations and modifications in their N-terminal membrane-spanning domains. Both of these were expressed in Escherichia coli at levels of 500 nmol/l. The protein variants were purified and tested for the solubility in the absence of detergent. Variant bCYP51-d1 exhibited ∼10-fold better solubility over variant bCYT51-d2. The bCYP51-d1 eluted as a single peak in size-exclusion chromatography, corresponding to its monomeric form. The activity of bCYP51-d1 is similar to that of recombinant human CYP51 with a non-truncated membrane-spanning region. High solubility and low tendency to non-specific oligomer formation make bCYP51-d1 a promising candidate for successful crystallization, which may finally allow the structural determination of this important mammalian enzyme.     

A relationship between the activities of hepatic lanosterol 14 alpha-demethylase and 3-hydroxy-3-methylglutaryl-CoA reductase.

At 1-2 h after intragastric administration of ketoconazole, a cytochrome P-450 inhibitor, to rats, there was a 50-60% decrease in the activity of hepatic 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase. Inhibition reached a maximum at 6-12 h after the drug was given, but after 24 h enzyme activity was stimulated by 60%. The rates of synthesis of hepatic non-saponifiable lipids in vivo showed a similar time-dependent pattern of change. During the first few hours after drug administration, the hepatic cytochrome P-450-dependent metabolism of lanosterol was suppressed in vivo. However, 24 h after treatment, this activity was stimulated, an effect which was also observed by pre-treatment of the rats with the drug for several days. Suppression of hepatic HMG-CoA reductase and lanosterol 14 alpha-demethylase activities was accompanied by a relative increase in the accumulation of labelled polar sterols in the liver in vivo. In the intestine, ketoconazole also resulted in a rapid decline in the rate of synthesis of non-saponifiable lipids and an inhibition of lanosterol 14 alpha-demethylation in vivo. However, in contrast with the liver, there was no stimulation of non-saponifiable lipid synthesis after 24 h.     

Homology modeling of lanosterol 14alpha-demethylase of Candida albicans and Aspergillus fumigatus and insights into the enzyme-substrate Interactions

The crystal structure of 14alpha-sterol demethylase from Mycobacterium tuberculosis (MT_14DM) provides a good template for modeling the three dimensional structure of lanosterol 14alpha-demethylase, which is the target of azole antifungal agents. Homologous 3D models of lanosterol 14alpha-demethylase from Candida albicans (CA_14DM) and Aspergillus fumigatus (AF_14DM) were built on the basis of the crystal coordinates of MT_14DM in complex with 4-phenylimidazole and fluconazole. The reliability of the two models was assessed by Ramachandran plots, Profile-3D analysis, and by analyzing the consistency of the two models with the experimental data on the P450(14DM). The overall structures of the resulting CA_14DM model and AF_14DM model are similar to those of the template structures. The two models remain the core structure characteristic for cytochrome P450s and most of the insertions and deletions expose the molecular surface. The structurally and functionally important residues such as the heme binding residues, the residues lining the substrate access channel, and residues in active site were identified from the model. To explore the binding mode of the substrate with the two models, 24(28)-methylene-24,25-dihydrolanosterol was docked into the active site of the two models and hydrophobic interaction and hydrogen-bonding were found to play an important role in substrate recognition and orientation. These results provided a basis for experiments to probe structure-function relationships in the P450(14DM). Although CA_14DM and AF_14DM shared similar core structural character, the active site of the two models were quite different, thus allowing the rational design of specific inhibitors to the target enzyme and the discovery of novel antifungal agents with broad spectrum.     

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.     

The Saccharomyces cerevisiae Cdc14 phosphatase is implicated in the structural organization of the nucleolus

Cdc14, a dual-specificity protein phosphatase, has been previously implicated in triggering exit from mitosis in the yeast Saccharomyces cerevisiae. Using immunofluorescence microscopy and immunogold labeling, we demonstrate that a functional HA-tagged version of the phosphatase Cdc14 localizes to the nucleolus. Moreover, Cdc14-HA co-localized with the nucleolar NOP2 and GAR1 proteins. By immunofluorescence, Cdc14-HA was found in the nucleolus during most of the mitotic cell cycle, except during anaphase-telophase when it redistributed along the mitotic spindle. While this work was in progress, the same pattern of Cdc14 localization was described by others (Visintin et al, Nature 398 (1999) 818). Constitutive overexpression of CDC14 was toxic and led to cell cycle arrest of cells, mainly in G1. This correlated with the appearance of abnormal nuclear structures. A genetic search for suppressors of the lethality associated with CDC14 overexpression identified YJL076W. Because overproduction of Yj1076w buffered the toxic effect of Cdc14 overproduction, this suggested that it might be a substrate of Cdc14. This has indeed been found to be the case by others who recently described Yj1076w/Netl as a nucleolar protein that physically associates with Cdc14 (Shou et al, Cell 97 (1999) 233). The present data confirm several recently uncovered aspects of the regulation of Cdc14 localization and activity and suggest that the level of expression of CDC14 influences the structural organization of the nucleolus.     

A possible role for cytochrome P-450 during the biosynthesis of zymosterol from lanosterol by Saccharomyces cerevisiae

A cell-free system has been obtained from $\text{Saccharomyces cerevisiae}$ which is capable of efficiently converting lanosterol¹ to a mixture of 4-demethyl sterols, quantitatively the most important identifiable component of which was zymosterol. Little or no ergosterol was synthesized. In the presence of carbon monoxide, the rate of zymosterol biosynthesis from lanosterol was decreased by 57% compared with that observed in control incubations and the amount of unmetabolized lanosterol was greater. Mitochondrial electron transport inhibitors such as cyanide and antimycin A had no effect on the overall rate of 4-demethyl sterol biosynthesis from lanosterol nor on the degree of inhibition by carbon monoxide.