Molecular diversity of sterol 14α‐demethylase substrates in plants, fungi and humans

Lamb, David C.; Kelly, Diane; Kelly, Steven L.

doi:10.1016/s0014-5793(98)00247-6

Cited by 68 publications

(53 citation statements)

References 16 publications

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“…This compound was identified as the silylated 14␣-demethylated 4␣-methyl-5␣-ergosta-8,14,24(28)-trien-3␤-ol. In control experiments, the fragmentation pattern of the molecular ion of the silylated product catalyzed by MCCYP51FX is identical to the mass spectrometry fragmentation pattern observed for the 14␣-demethylated product from lanosterol generated in a purified, reconstituted C. albicans CYP51/NADPH cytochrome P450 reductase enzyme system (28). The activity was calculated to be 0.24 nmol of demethylated product formed per minute per nanomole of MCCYP51FX, with no metabolism detected in the absence of MCCYP51FX or NADPH.…”

Section: Protein Combinationsupporting

confidence: 52%

A Novel Sterol 14α-Demethylase/Ferredoxin Fusion Protein (MCCYP51FX) from Methylococcus capsulatusRepresents a New Class of the Cytochrome P450 Superfamily

Jackson

Lamb

Marczylo

et al. 2002

Journal of Biological Chemistry

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Sterol 14␣-demethylase encoded by CYP51 is a member of the cytochrome P450 (CYP) superfamily of enzymes and has been shown to have an essential role in sterol biosynthesis in eukaryotes, with orthologues recently being described in some bacteria. Examination of the genome sequence data for the proteobacterium Methylococcus capsulatus, a bacterial species known to produce sterol, revealed the presence of a single CYP with strong homology to CYP51, particularly to a form in Mycobacterium tuberculosis. This M. capsulatus CYP51 protein represents a new class of CYP consisting of the CYP domain naturally fused to a ferredoxin domain at the C terminus via an alanine-rich linker. Expression of the M. capsulatus MCCYP51FX fusion in Escherichia coli yielded a P450, which, when purified to homogeneity, had the predicted molecular mass ϳ62 kDa on SDS/ PAGE and bound lanosterol as a putative substrate. Sterol 14␣-demethylase activity was shown (0.24 nmol of lanosterol metabolized per minute per nanomole of MCCYP51FX fusion) by gas chromatography/mass spectrometry with the activity dependent upon the presence of ferredoxin reductase and NADPH. Our unique findings describe a new class of naturally existing cytochrome P450, which will provide pivotal information for CYP structure/function in general. Cytochrome P450 (CYP)1 genes are members of one of the most functionally versatile superfamilies of enzymes found in nature. CYP enzymes are involved both in essential endogenous biosynthetic functions, e.g. steroids, hormones, and fatty acids, and in the metabolism of xenobiotic chemicals, e.g. drugs, carcinogens, and pollutants (1-3). The biodiversity of cytochrome P450 genes has been revealed in dramatic fashion by genome sequencing projects, and so much so that many thousands of different forms have been or will be identified shortly. Within bacteria, some species such as Escherichia coli lack cytochrome P450 genes, whereas others contain many, such as the 20 forms of Mycobacterium tuberculosis (4). Recently, genomic information on the methane-utilizing bacterium Methylococcus capsulatus has become available (www.tigr.org). This bacterium is one of the first and best elucidated of any prokaryotes for the production of sterols (5, 6), a feature generally associated with eukaryotes. These investigations used minimal sterol-free media for growth, and gas chromatography/mass spectrometry plus NMR for identification of 4-dimethyl, 4-methyl, and 4-demethyl sterols. This information indicated that a sterol 14-␣ demethylase was present in the organism that is encoded by a cytochrome P450, CYP51.The CYP-catalyzed reaction involves the molecular splitting of atmospheric oxygen to produce a molecule of water and a monooxygenated substrate (7). Two reducing equivalents are essential for this reaction. They are supplied by NADH or NADPH, depending on the origin of the CYP, and are transferred in two stages through either one or two redox partners. Different CYPs of eukaryotes and prokaryotes differ in their reductase partners as well as in...

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Section: Protein Combinationsupporting

confidence: 52%

A Novel Sterol 14α-Demethylase/Ferredoxin Fusion Protein (MCCYP51FX) from Methylococcus capsulatusRepresents a New Class of the Cytochrome P450 Superfamily

Jackson

Lamb

Marczylo

et al. 2002

Journal of Biological Chemistry

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“…In humans there are three CYP51 genes, including two pseudogenes and a functional gene on chromosome 7 . Substrates for CYP51 from mammals, plants, and fungi are lanosterol, obtusifoliol, and 24-methylene-dihydroxylanosterol, respectively (Lamb et al, 1998). Such substrate specificities can be explained by a limited number of amino acid substitutions in substrate recognition sites 1, 2, and 5 between mammalian and fungal CYP51, and it is likely that these locations are responsible for conferring selectivity for sterol metabolism (Yoshida et al, 1997).…”

mentioning

confidence: 99%

Three-Dimensional Quantitative Structure-Activity Relationship Analysis of Human CYP51 Inhibitors

Ekins¹,

Mankowski²,

Hoover³

et al. 2006

Drug Metab Dispos

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ABSTRACT:CYP51 fulfills an essential requirement for all cells, by catalyzing three sequential mono-oxidations within the cholesterol biosynthesis cascade. Inhibition of fungal CYP51 is used as a therapy for treating fungal infections, whereas inhibition of human CYP51 has been considered as a pharmacological approach to treat dyslipidemia and some forms of cancer. This study has demonstrated the potential for ligand-based computational pharmacophore modeling of human CYP51 and enables a high-throughput screening system for drug discovery and data base mining.

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“…Although CYP51 proteins isolated from bacteria through mammals have highly conserved regions, significant structural variability occurs in regions of the enzyme associated with the binding of sterol substrates and azole inhibitors (34,35). For example, CYP51 from human and Candida albicans both catalyze the demethylation of lanosterol, but with significantly different enzymatic activity (36). In contrast, CYP51 from Sorghum bicolor has a strict substrate specificity and selectivity for obtusifoliol, and cannot catalyze the demethylation of lanosterol (37).…”

Section: Discussionmentioning

confidence: 99%

Cloning and Characterization of CYP51 from Mycobacterium avium

Pietila

Vohra

Sanyal

et al. 2006

Am J Respir Cell Mol Biol

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Mycobacterium avium complex (MAC) causes chronic lung disease in immunocompetent people and disseminated infection in patients with AIDS. MAC is intrinsically resistant to many conventional antimycobacterial agents, it develops drug resistance rapidly to macrolide antibiotics, and patients with MAC infection experience frequent relapses or the inability to completely eradicate the infection with current treatment. Treatment regimens are prolonged and complicated by drug toxicity or intolerances. We sought to identify biochemical pathways in MAC that can serve as targets for novel antimycobacterial treatment. The cytochrome P450 enzyme, CYP51, catalyzes an essential early step in sterol metabolism, removing a methyl group from lanosterol in animals and fungi, or from obtusifoliol in plants. Azoles inhibit CYP51 function, leading to an accumulation of methylated sterol precursors. This perturbation of normal sterol metabolism compromises cell membrane integrity, resulting in growth inhibition or cell death. We have cloned and characterized a CYP51 from MAC that functions as a lanosterol 14␣-demethylase. We show the direct interactions of azoles with purified MAC-CYP51 by absorbance and electron paramagnetic resonance spectroscopy, and determine the minimum inhibitory concentrations (MICs) of econazole, ketoconazole, itraconazole, fluconazole, and voriconazole against MAC. Furthermore, we demonstrate that econazole has a MIC of 4 g/ml and a minimum bacteriocidal concentration of 4 g/ml, whereas ketoconazole has a MIC of 8 g/ml and a minimum bacteriocidal concentration of 16 g/ml. Itraconazole, voriconazole, and fluconazole did not inhibit MAC growth to any significant extent.

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Molecular diversity of sterol 14α‐demethylase substrates in plants, fungi and humans

Cited by 68 publications

References 16 publications

A Novel Sterol 14α-Demethylase/Ferredoxin Fusion Protein (MCCYP51FX) from Methylococcus capsulatusRepresents a New Class of the Cytochrome P450 Superfamily

A Novel Sterol 14α-Demethylase/Ferredoxin Fusion Protein (MCCYP51FX) from Methylococcus capsulatusRepresents a New Class of the Cytochrome P450 Superfamily

Three-Dimensional Quantitative Structure-Activity Relationship Analysis of Human CYP51 Inhibitors

Cloning and Characterization of CYP51 from Mycobacterium avium

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