Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Δ7-24, 25-dihydrolanosterol

Hargrove, Tatiana Y.; Wawrzak, Z.; Liu, Jialin; Waterman, Michael R.; Nes, W. David; Lepesheva, Galina I.

doi:10.1194/jlr.m021865

Cited by 63 publications

(87 citation statements)

References 54 publications

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“…Minimally biased Fo-Fc electron density omit maps, calculated with lanosterol omitted to place the lanosterol correctly, resulted in a ligand placement with reasonable correlation coefficient (0.813). Bound lanosterol is oriented differently from other lanosterol-like molecules in related CYP51s (24), and the electron density offers no evidence of these alternate orientations.…”

Section: Resultsmentioning

confidence: 99%

“…The crystal structures also locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding the drug resistance associated with orthologs of the enzyme found in fungal pathogens. several inhibitors, including ketoconazole, posaconazole, FLC, and substrate analogs such as 14α-methylenecyclopropyl-Δ7-24,25-dihydrolanosterol, have been visualized (19,20,22,24).…”

Section: Significancementioning

confidence: 99%

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Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Monk

Tomasiak

Keniya

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

248

283

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Bitopic integral membrane proteins with a single transmembrane helix play diverse roles in catalysis, cell signaling, and morphogenesis. Complete monospanning protein structures are needed to show how interaction between the transmembrane helix and catalytic domain might influence association with the membrane and function. We report crystal structures of full-length Saccharomyces cerevisiae lanosterol 14α-demethylase, a membrane monospanning cytochrome P450 of the CYP51 family that catalyzes the first postcyclization step in ergosterol biosynthesis and is inhibited by triazole drugs. The structures reveal a well-ordered N-terminal amphipathic helix preceding a putative transmembrane helix that would constrain the catalytic domain orientation to lie partly in the lipid bilayer. The structures locate the substrate lanosterol, identify putative substrate and product channels, and reveal constrained interactions with triazole antifungal drugs that are important for drug design and understanding drug resistance.M embrane proteins that span the lipid bilayer once constitute around 50% of all integral membrane proteins (1). Although monospanning membrane proteins carry out numerous key biological functions, including environmental sensing, organellespecific catalysis, and the regulation of cell morphology, only individual domains or subdomains are currently represented in the Protein Data Bank, and structural information about interactions between their transmembrane domains and extramembranous components is lacking. Cytochrome P450 proteins are prominent enzymes with orthologs found in all kingdoms of life. In eukaryotes, microsomal members of this major family of mixed-function mono-oxygenases contain a single transmembrane helix and can be grouped in two broad functional categories: biodefense, such as the first phase of xenobiotic detoxification, and core metabolism including reactions in sterol biosynthesis and fatty acid oxidation (2).The lanosterol 14α-demethylases or CYP51 enzymes, probably the most genetically ancient of the cytochrome P450 families, play a central role in cholesterol or ergosterol biosynthesis (3). CYP51s carry out three consecutive mono-oxygenase reaction cycles to remove the 14α-methyl group from lanosterol to yield 4,4-dimethyl-cholesta-8,14,24-trienol, a key precursor in cholesterol and ergosterol biosynthesis, releasing water and formic acid (3). Because of the key roles that CYP51s play in yeast, filamentous fungi, and some parasitic protozoa, these enzymes are therapeutic targets for antimicrobial agents, including fluconazole (FLC), voriconazole (VCZ), and itraconazole (ITC) (4). Fungal infections play an increasingly significant role in disease, impacting agriculture ecosystems and human health, especially in immunocompromised individuals (5-7) for whom antifungal resistance continually poses a threat (8). In humans CYP51 is being tested as a target for cholesterol-lowering drugs (9) and in antiangiogenic cancer therapies (10). A limited set of cytochrome P450 isoforms (1A2, 2C8, 2C...

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Section: Resultsmentioning

confidence: 99%

Section: Significancementioning

confidence: 99%

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Monk

Tomasiak

Keniya

et al. 2014

Proc. Natl. Acad. Sci. U.S.A.

248

283

View full text Add to dashboard Cite

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“…However, when AZA was combined with itraconzaole at their ED 50 , cell proliferation stopped and cells died. The total dependence of inhibitor-treated cells on ergosterol for sparking cell proliferation is thus understandable and can be reconciled with all experimental observations, by their depletion of ergosterol production, and may turn out to be useful in the design of a new generation of irreversible suicide substrates targeted to impair essential sterol functions in trypanosomes ( 8,57 ). All things considered, due to the limits of monotherapies, greater emphasis should be placed on orally administered analogs of Tb SMT catalysis in combination with specifi c azoles, which together might be effective in smaller amounts and have less deleterious effects on the host than the drugs presently in use.…”

Section: Discussionmentioning

confidence: 99%

Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth

Haubrich¹,

Singha²,

Miller³

et al. 2015

Journal of Lipid Research

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Abbreviations: AZA, 25-azalanosterol; BSF, bloodstream form; DOX, doxycycline; ED, effective dose; FGM, full-growth medium; ITC, itraconazole; LDM, lipid-depleted medium; PCF, procyclic form; RRTc, relative retention time with cholesterol used as standard; SAM, S-adenosyl-lmethionine; SDM, sterol C14-demethylase; SMT, sterol C24-methyltransferase; Tb SDM, Trypanosoma brucei analyzed by RNAi silencing and inhibition of sterol C14-demethylase; Tb SMT, Trypanosoma brucei analyzed by RNAi silencing and inhibition of sterol C24-methyltransferase; TetR, tetracycline repressor .

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“…Y103 and F110 (B= helix) are invariant in the whole family (Ͼ300 sequenced proteins), and Y116 (BЉ helix) is conserved in vertebrates, fungi, and protozoa yet is replaced by F in all plants. In the ligand-free and sterol-bound protozoan CYP51 structures (PDB accession numbers 3G1Q [48] and 3P99 [49]), Y103 and Y116 form hydrogen bonds with the heme ring A and D propionates, respectively. The binding of some heme-coordinating ligands, however, was found to disrupt these H bonds (32,33,36,48,50), which is likely to enhance the inhibitory potency of the compounds by weakening the P450 heme support from the protein moiety (51).…”

Section: Resultsmentioning

confidence: 99%

Clinical Candidate VT-1161's Antiparasitic Effect In Vitro , Activity in a Murine Model of Chagas Disease, and Structural Characterization in Complex with the Target Enzyme CYP51 from Trypanosoma cruzi

Hoekstra

Hargrove

Wawrzak

et al. 2016

Antimicrob Agents Chemother

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Structural complex of sterol 14α-demethylase (CYP51) with 14α-methylenecyclopropyl-Δ7-24, 25-dihydrolanosterol

Cited by 63 publications

References 54 publications

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Architecture of a single membrane spanning cytochrome P450 suggests constraints that orient the catalytic domain relative to a bilayer

Discovery of an ergosterol-signaling factor that regulates Trypanosoma brucei growth

Clinical Candidate VT-1161's Antiparasitic Effect In Vitro , Activity in a Murine Model of Chagas Disease, and Structural Characterization in Complex with the Target Enzyme CYP51 from Trypanosoma cruzi

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