Publications

• Obtusifoliol 14alpha-demethylase (CYP51) antisense Arabidopsis shows slow growth and long life.

  Kushiro M., Nakano T., Sato K., Yamagishi K., Asami T., Nakano A., Takatsuto S., Fujioka S., Ebizuka Y., Yoshida S.

  Obtusifoliol 14alpha-demethylase is a plant orthologue of sterol 14alpha-demethylase (CYP51) essential in sterol biosynthesis. We have prepared CYP51 antisense Arabidopsis in order to shed light on the sterol and steroid hormone biosynthesis in plants. Arabidopsis putative CYP51 cDNA (AtCYP51) was ... >> More

  Obtusifoliol 14alpha-demethylase is a plant orthologue of sterol 14alpha-demethylase (CYP51) essential in sterol biosynthesis. We have prepared CYP51 antisense Arabidopsis in order to shed light on the sterol and steroid hormone biosynthesis in plants. Arabidopsis putative CYP51 cDNA (AtCYP51) was obtained from Arabidopsis expressed sequence tag (EST) library and its function was examined in a yeast lanosterol 14alpha-demethylase (Erg11) deficient mutant. A recombinant AtCYP51 protein fused with a yeast Erg11 signal-anchor peptide was able to complement the erg11 mutation, which confirmed AtCYP51 to be a functional sterol 14alpha-demethylase. AtCYP51 was then used to generate transgenic Arabidopsis by transforming with pBI vector harboring AtCYP51 in the antisense direction under CaMV35S promoter. The resulting transgenic plants were decreased in accumulation of AtCYP51 mRNA and increased in the amount of endogenous obtusifoliol. They showed a semidwarf phenotype in the early growth stage and a longer life span than control plants. This newly found phenotype is different from previously characterized brassinosteroid (BR)-deficient campesterol biosynthesis mutants. << Less

  Biochem. Biophys. Res. Commun. 285:98-104(2001) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Structural basis of human CYP51 inhibition by antifungal azoles.

  Strushkevich N., Usanov S.A., Park H.W.

  The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, m ... >> More

  The obligatory step in sterol biosynthesis in eukaryotes is demethylation of sterol precursors at the C14-position, which is catalyzed by CYP51 (sterol 14-alpha demethylase) in three sequential reactions. In mammals, the final product of the pathway is cholesterol, while important intermediates, meiosis-activating sterols, are produced by CYP51. Three crystal structures of human CYP51, ligand-free and complexed with antifungal drugs ketoconazole and econazole, were determined, allowing analysis of the molecular basis for functional conservation within the CYP51 family. Azole binding occurs mostly through hydrophobic interactions with conservative residues of the active site. The substantial conformational changes in the B' helix and F-G loop regions are induced upon ligand binding, consistent with the membrane nature of the protein and its substrate. The access channel is typical for mammalian sterol-metabolizing P450 enzymes, but is different from that observed in Mycobacterium tuberculosis CYP51. Comparison of the azole-bound structures provides insight into the relative binding affinities of human and bacterial P450 enzymes to ketoconazole and fluconazole, which can be useful for the rational design of antifungal compounds and specific modulators of human CYP51. << Less

  J. Mol. Biol. 397:1067-1078(2010) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Arabidopsis cyp51 mutant shows postembryonic seedling lethality associated with lack of membrane integrity.

  Kim H.B., Schaller H., Goh C.H., Kwon M., Choe S., An C.S., Durst F., Feldmann K.A., Feyereisen R.

  CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14alpha-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes ... >> More

  CYP51 exists in all organisms that synthesize sterols de novo. Plant CYP51 encodes an obtusifoliol 14alpha-demethylase involved in the postsqualene sterol biosynthetic pathway. According to the current gene annotation, the Arabidopsis (Arabidopsis thaliana) genome contains two putative CYP51 genes, CYP51A1 and CYP51A2. Our studies revealed that CYP51A1 should be considered an expressed pseudogene. To study the functional importance of the CYP51A2 gene in plant growth and development, we isolated T-DNA knockout alleles for CYP51A2. Loss-of-function mutants for CYP51A2 showed multiple defects, such as stunted hypocotyls, short roots, reduced cell elongation, and seedling lethality. In contrast to other sterol mutants, such as fk/hydra2 and hydra1, the cyp51A2 mutant has only minor defects in early embryogenesis. Measurements of endogenous sterol levels in the cyp51A2 mutant revealed that it accumulates obtusifoliol, the substrate of CYP51, and a high proportion of 14alpha-methyl-delta8-sterols, at the expense of campesterol and sitosterol. The cyp51A2 mutants have defects in membrane integrity and hypocotyl elongation. The defect in hypocotyl elongation was not rescued by the exogenous application of brassinolide, although the brassinosteroid-signaling cascade is apparently not affected in the mutants. Developmental defects in the cyp51A2 mutant were completely rescued by the ectopic expression of CYP51A2. Taken together, our results demonstrate that the Arabidopsis CYP51A2 gene encodes a functional obtusifoliol 14alpha-demethylase enzyme and plays an essential role in controlling plant growth and development by a sterol-specific pathway. << Less

  Plant Physiol. 138:2033-2047(2005) [PubMed] [EuropePMC]

• The ubiquitously expressed human CYP51 encodes lanosterol 14 alpha-demethylase, a cytochrome P450 whose expression is regulated by oxysterols.

  Stroemstedt M., Rozman D., Waterman M.R.

  Sterol biosynthesis requires the removal of the 14 alpha-methyl group from lanosterol in animals and fungi and from obtusifoliol in plants. This reaction is catalyzed by a microsomal cytochrome P450, the sterol 14 alpha-demethylase (P450(14DM), which is the only P450 described so far to be express ... >> More

  Sterol biosynthesis requires the removal of the 14 alpha-methyl group from lanosterol in animals and fungi and from obtusifoliol in plants. This reaction is catalyzed by a microsomal cytochrome P450, the sterol 14 alpha-demethylase (P450(14DM), which is the only P450 described so far to be expressed in different phyla. A cDNA encoding human P450(14DM) was isolated from a liver cDNA library using a partial rat lanosterol 14 alpha-demethylase cDNA probe. The deduced amino acid sequence is 93% and 38--42% identical to rat and fungal P450(14DM), respectively. Expression of the human CYP51 cDNA in Escherichia coli showed that the cDNA encodes an enzyme having lanosterol 14 alpha-demethylase activity. Northern blot analysis showed that CYP51 mRNA is ubiquitously expressed with highest levels in testis, ovary, adrenal, prostate, liver, kidney, and lung. Many genes involved in cholesterol homeostasis are regulated by cholesterol or its metabolites. In the case of CYP51, cholesterol deprivation led to a 2.6-to 3.8-fold induction of mRNA levels in human adrenocortical H295R cells and this effect was suppressed by the addition of 25-hydroxycholesterol. In human hepatoma HepG2 cells, no effect of cholesterol deprivation was observed; however, the levels of CYP51 mRNA were reduced 4-to 6-fold by the addition of 25-hydroxycholesterol. Thus, like several other genes in the cholesterol biosynthetic pathway, including the 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase, HMG CoA reductase, squalene synthase, and farnesyl diphosphate synthase, the expression of the human CYP51 is suppressed by oxysterols. << Less

  Arch. Biochem. Biophys. 329:73-81(1996) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• CYP51 from Trypanosoma cruzi: a phyla-specific residue in the B' helix defines substrate preferences of sterol 14alpha-demethylase.

  Lepesheva G.I., Zaitseva N.G., Nes W.D., Zhou W., Arase M., Liu J., Hill G.C., Waterman M.R.

  A potential drug target for treatment of Chagas disease, sterol 14alpha-demethylase from Trypanosoma cruzi (TCCYP51), was found to be catalytically closely related to animal/fungi-like CYP51. Contrary to the ortholog from Trypanosoma brucei (TB), which like plant CYP51 requires C4-monomethylated s ... >> More

  A potential drug target for treatment of Chagas disease, sterol 14alpha-demethylase from Trypanosoma cruzi (TCCYP51), was found to be catalytically closely related to animal/fungi-like CYP51. Contrary to the ortholog from Trypanosoma brucei (TB), which like plant CYP51 requires C4-monomethylated sterol substrates, TCCYP51 prefers C4-dimethylsterols. Sixty-six CYP51 sequences are known from bacteria to human, their sequence homology ranging from approximately 25% between phyla to approximately 80% within a phylum. TC versus TB is the first example of two organisms from the same phylum, in which CYP51s (83% amino acid identity) have such profound differences in substrate specificity. Substitution of animal/fungi-like Ile105 in the B' helix to Phe, the residue found in this position in all plant and the other six CYP51 sequences from Trypanosomatidae, dramatically alters substrate preferences of TCCYP51, converting it into a more plant-like enzyme. The rates of 14alpha-demethylation of obtusifoliol and its 24-demethyl analog 4alpha-,4alpha-dimethylcholesta-8,24-dien-3beta-ol(norlanosterol) increase 60- and 150-fold, respectively. Turnover of the three 4,4-dimethylated sterol substrates is reduced approximately 3.5-fold. These catalytic properties correlate with the sterol binding parameters, suggesting that Phe in this position provides necessary interactions with C4-monomethylated substrates, which Ile cannot. The CYP51 substrate preferences imply differences in the post-squalene portion of sterol biosynthesis in TC and TB. The phyla-specific residue can be used to predict preferred substrates of new CYP51 sequences and subsequently for the development of new artificial substrate analogs, which might serve as highly specific inhibitors able to kill human parasites. << Less

  J. Biol. Chem. 281:3577-3585(2006) [PubMed] [EuropePMC]

  This publication is cited by 3 other entries.

• Identification, modeling and ligand affinity of early deuterostome CYP51s, and functional characterization of recombinant zebrafish sterol 14alpha-demethylase.

  Morrison A.M., Goldstone J.V., Lamb D.C., Kubota A., Lemaire B., Stegeman J.J.

  <h4>Background</h4>Sterol 14α-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-ma ... >> More

  <h4>Background</h4>Sterol 14α-demethylase (cytochrome P450 51, CYP51, P45014DM) is a microsomal enzyme that in eukaryotes catalyzes formation of sterols essential for cell membrane function and as precursors in biosynthesis of steroid hormones. Functional properties of CYP51s are unknown in non-mammalian deuterostomes.<h4>Methods</h4>PCR-cloning and sequencing and computational analyses (homology modeling and docking) addressed CYP51 in zebrafish Danio rerio, the reef fish sergeant major Abudefduf saxatilis, and the sea urchin Strongylocentrotus purpuratus. Following N-terminal amino acid modification, zebrafish CYP51 was expressed in Escherichia coli, and lanosterol 14α-demethylase activity and azole inhibition of CYP51 activity were characterized using GC-MS.<h4>Results</h4>Molecular phylogeny positioned S. purpuratus CYP51 at the base of the deuterostome clade. In zebrafish, CYP51 is expressed in all organs examined, most strongly in intestine. The recombinant protein bound lanosterol and catalyzed 14α-demethylase activity, at 3.2nmol/min/nmol CYP51. The binding of azoles to zebrafish CYP51 gave KS (dissociation constant) values of 0.26μM for ketoconazole and 0.64μM for propiconazole. Displacement of carbon monoxide also indicated zebrafish CYP51 has greater affinity for ketoconazole. Docking to homology models showed that lanosterol docks in fish and sea urchin CYP51s with an orientation essentially the same as in mammalian CYP51s. Docking of ketoconazole indicates it would inhibit fish and sea urchin CYP51s.<h4>Conclusions</h4>Biochemical and computational analyses are consistent with lanosterol being a substrate for early deuterostome CYP51s.<h4>General significance</h4>The results expand the phylogenetic view of animal CYP51, with evolutionary, environmental and therapeutic implications. << Less

  Biochim. Biophys. Acta 1840:1825-1836(2014) [PubMed] [EuropePMC]

• Cloning and functional expression of the cDNA encoding rat lanosterol 14-alpha demethylase.

  Sloane D.L., So O.Y., Leung R., Scarafia L.E., Saldou N., Jarnagin K., Swinney D.C.

  Lanosterol 14 alpha-demethylase (LDM) is a cytochrome P-450 enzyme in the biosynthetic pathway of cholesterol. As such, it represents a target for cholesterol-lowering drugs. Rat LDM (rLDM) has been purified from the livers of rats treated with cholestyramine. The purified protein was used to gene ... >> More

  Lanosterol 14 alpha-demethylase (LDM) is a cytochrome P-450 enzyme in the biosynthetic pathway of cholesterol. As such, it represents a target for cholesterol-lowering drugs. Rat LDM (rLDM) has been purified from the livers of rats treated with cholestyramine. The purified protein was used to generate tryptic fragments which were then sequenced. The amino acid (aa) sequences were used to design oligodeoxyribonucleotide primers and a DNA fragment was generated by RT-PCR to probe a phagemid library. A clone encoding rLDM was isolated from the livers of cholestyramine-treated rats. The clone contains an open reading frame encoding a polypeptide of 486 aa and a predicted molecular mass of 55 045 Da. The deduced aa sequence shows a high degree of identity to the yeast LDM sequences, as well as sequences which match typical P-450 sequence motifs. When produced in a baculovirus/insect cell culture system, LDM activity was detected and inhibited by the specific inhibitor azalanstat with an IC50 value of less than 2 nM. The isolation of this full-length coding sequence should facilitate research into understanding the direct and indirect effects of LDM in the regulation of cholesterol biosynthesis and the search for cholesterol-lowering drugs. << Less

  Gene 161:243-248(1995) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.