Structural Basis of Human CYP51 Inhibition by Antifungal Azoles

Strushkevich, Natallia; Usanov, Sergey A.; Park, Hee-Won

doi:10.1016/j.jmb.2010.01.075

Cited by 237 publications

(214 citation statements)

References 54 publications

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“…We used molecular modeling and docking approaches to gain further insights into the potential reaction mechanism of AsCYP51H10, this time building a homology model based on the crystal structure of human CYP51 in complex with ketoconazole (36). Human CYP51 has a higher sequence identity to AsCYP51H10 (34%) than does MtCYP51B1 (28%) and so represents a more appropriate template.…”

Section: Resultsmentioning

confidence: 99%

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

Geisler

Hughes

Sainsbury

et al. 2013

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

151

134

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Members of the cytochromes P450 superfamily (P450s) catalyze a huge variety of oxidation reactions in microbes and higher organisms. Most P450 families are highly divergent, but in contrast the cytochrome P450 14α-sterol demethylase (CYP51) family is one of the most ancient and conserved, catalyzing sterol 14α-demethylase reactions required for essential sterol synthesis across the fungal, animal, and plant kingdoms. Oats (Avena spp.) produce antimicrobial compounds, avenacins, that provide protection against disease. Avenacins are synthesized from the simple triterpene, β-amyrin. Previously we identified a gene encoding a member of the CYP51 family of cytochromes P450, AsCyp51H10 (also known as Saponin-deficient 2, Sad2), that is required for avenacin synthesis in a forward screen for avenacin-deficient oat mutants. sad2 mutants accumulate β-amyrin, suggesting that they are blocked early in the pathway. Here, using a transient plant expression system, we show that AsCYP51H10 is a multifunctional P450 capable of modifying both the C and D rings of the pentacyclic triterpene scaffold to give 12,13β-epoxy-3β,16β-dihydroxy-oleanane (12,13β-epoxy-16β-hydroxy-β-amyrin). Molecular modeling and docking experiments indicate that C16 hydroxylation is likely to precede C12,13 epoxidation. Our computational modeling, in combination with analysis of a suite of sad2 mutants, provides insights into the unusual catalytic behavior of AsCYP51H10 and its active site mutants. Fungal bioassays show that the C12,13 epoxy group is an important determinant of antifungal activity. Accordingly, the oat AsCYP51H10 enzyme has been recruited from primary metabolism and has acquired a different function compared to other characterized members of the plant CYP51 family-as a multifunctional stereo-and regio-specific hydroxylase in plant specialized metabolism.

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Section: Resultsmentioning

confidence: 99%

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

Geisler

Hughes

Sainsbury

et al. 2013

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

151

134

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“…Data were processed, integrated, and scaled using the HKL2000 (53) package. An initial model was determined by molecular replacement using the soluble domain of the HsCYP51 model 3LD6 (20) in Phaser (54). Refinement was performed using both Refmac5 in the CCP4 (55) suite of programs and Phenix (56), and model building was performed using Coot (57).…”

Section: Methodsmentioning

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%

“…For the eukaryotic cytochrome P450 enzymes that contain transmembrane segments, most X-ray structures have been obtained after removal of the N-terminal transmembrane domain to facilitate expression and crystallization (13)(14)(15)(16)(17)(18), as is the case with many other monospanning membrane protein structures. Resolved CYP51 structures include the soluble enzyme from Mycobacterium tuberculosis (19) and N-terminal truncated enzymes from Homo sapiens (20), Trypanosoma cruzi (21), Trypanosoma brucei (22), and Leishmania infantum (23). The available eukaryotic cytochrome P450 structures show the catalytic subunit has a conserved fold surrounding a central prosthetic heme group.…”

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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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“…Human 51A catalyzes the 14␣-demethylation of lanosterol, another carbon-carbon bond scission reaction, in the pathway for de novo synthesis of cholesterol. It is anticipated that the availability of structures for human 51A1 (43) and 51A1 orthologs in fungal pathogens (44 -46) will aid in the design of drugs that are more selective for fungal 51A relative to the human enzyme.…”

Section: Specialist Enzymesmentioning

confidence: 99%

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

Johnson¹,

Stout

2013

Journal of Biological Chemistry

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X-ray crystal structures are available for 29 eukaryotic microsomal, chloroplast, or mitochondrial cytochrome P450s, including two non-monooxygenase P450s. These structures provide a basis for understanding structure-function relations that underlie their distinct catalytic activities. Moreover, structural plasticity has been characterized for individual P450s that aids in understanding substrate binding in P450s that mediate drug clearance.

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Structural Basis of Human CYP51 Inhibition by Antifungal Azoles

Cited by 237 publications

References 54 publications

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

Biochemical analysis of a multifunctional cytochrome P450 (CYP51) enzyme required for synthesis of antimicrobial triterpenes in plants

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

Structural Diversity of Eukaryotic Membrane Cytochrome P450s

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