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The CYP51F1 Gene of Leptographium qinlingensis: Sequence Characteristic, Phylogeny and Transcript Levels.

Dai L, Li Z, Yu J, Ma M, Zhang R, Chen H, Pham T - Int J Mol Sci (2015)

Bottom Line: We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae).The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1) and transmembrane helix 1 (TMH1).The minimal inhibitory concentrations (MIC) of terpenoid and azole fungicides (itraconazole (ITC)) and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.

View Article: PubMed Central - PubMed

Affiliation: College of Forestry, Northwest A&F University, Yangling 712100, China. dailulu@nwafu.edu.cn.

ABSTRACT
Leptographium qinlingensis is a fungal associate of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandi) that must overcome the terpenoid oleoresin defenses of host trees. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include export and the use of these compounds as a carbon source. As one of the fungal cytochrome P450 proteins (CYPs), which play important roles in general metabolism, CYP51 (lanosterol 14-α demethylase) can catalyze the biosynthesis of ergosterol and is a target for antifungal drug. We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae). The transcription level of CYP51F1 following treatment with terpenes and pine phloem extracts was upregulated, while using monoterpenes as the only carbon source led to the downregulation of CYP5F1 expression. The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1) and transmembrane helix 1 (TMH1). The minimal inhibitory concentrations (MIC) of terpenoid and azole fungicides (itraconazole (ITC)) and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.

No MeSH data available.


Lanosterol and itraconazole (ITC) binding in CYP51F1. (A) Lanosterol is depicted with carbon atoms colored cyan and heme with carbon atoms colored yellow. Selected oxygen atoms are colored red; nitrogen is blue; and iron is stone blue; (B) ITC is depicted with carbon atoms colored green, and other atoms are colored as in (A). A 2D diagram of the interaction between the protein and ligands is shown below: (a) lanosterol and (b) ITC. Purple dot: amino acids for a hydrogen bond, static electricity and polarity interactions. Green dot: amino acids for VDW (Van der Waals) interactions. Blue shadow: solvent around the amino acids and atoms. Green arrow: electron donor for a hydrogen bond.
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ijms-16-12014-f007: Lanosterol and itraconazole (ITC) binding in CYP51F1. (A) Lanosterol is depicted with carbon atoms colored cyan and heme with carbon atoms colored yellow. Selected oxygen atoms are colored red; nitrogen is blue; and iron is stone blue; (B) ITC is depicted with carbon atoms colored green, and other atoms are colored as in (A). A 2D diagram of the interaction between the protein and ligands is shown below: (a) lanosterol and (b) ITC. Purple dot: amino acids for a hydrogen bond, static electricity and polarity interactions. Green dot: amino acids for VDW (Van der Waals) interactions. Blue shadow: solvent around the amino acids and atoms. Green arrow: electron donor for a hydrogen bond.

Mentions: The cavity volume at the binding sites was calculated, using DS (Binding Site module) for the CYP51F1 protein structure. A binding site with a maximum volume (x: 22.488, y: 8.09, z: 13.729; volume: 836.5) was selected manually, as it should have the appropriate volume for a molecule to adopt minimal energy. For monoterpenes (limonene, 3-carene and pinene) and sesquiterpenes (β-caryophyllene and longifolene), one pose of each was generated using “LibDock.” Seven DS scoring functions (Ligscore1, Ligscore2, -PLP1, -PLP2, Jain, -PMF and -PMF04) and consensus scoring functions were used to re-evaluate the position of docked molecules (Table 4). The position of terpenoid molecules were shown in the binding pocket of CYP51F1 colored green (Figure 6). The interactions of CYP51F1-terpenoid molecules were shown in a 2D diagram (Figure S1). For lanosterol and ITC, the most suitable docking mode for each molecule received a consensus score of seven and six, respectively. More than twelve amino acid residues participated in the interactions between CYP51F1 and the ligands lanosterol and ITC (Figure 7). Three (Ala 302, 306, Leu 303) and six (Ala 306, Met 70, His 373, Gly 69, Ser 374, Hem 601) amino acid residues formed hydrogen bonds, static electricity and polarity interactions between CYP51F1 and the ligands lanosterol and ITC, respectively (Figure 7). These sixteen amino acid residues (Ala 507, Val 135, Phe 130, 229, 234, Met 376, Thr 126, Leu 509, Tyr 68, 122, 136, 506, Pro 231, Ile 372, 375 and Ser 508) formed Van der Waals (VDW) interactions in both CYP51F1-lanosterol and CYP51F1-ITC (Figure S2).


The CYP51F1 Gene of Leptographium qinlingensis: Sequence Characteristic, Phylogeny and Transcript Levels.

Dai L, Li Z, Yu J, Ma M, Zhang R, Chen H, Pham T - Int J Mol Sci (2015)

Lanosterol and itraconazole (ITC) binding in CYP51F1. (A) Lanosterol is depicted with carbon atoms colored cyan and heme with carbon atoms colored yellow. Selected oxygen atoms are colored red; nitrogen is blue; and iron is stone blue; (B) ITC is depicted with carbon atoms colored green, and other atoms are colored as in (A). A 2D diagram of the interaction between the protein and ligands is shown below: (a) lanosterol and (b) ITC. Purple dot: amino acids for a hydrogen bond, static electricity and polarity interactions. Green dot: amino acids for VDW (Van der Waals) interactions. Blue shadow: solvent around the amino acids and atoms. Green arrow: electron donor for a hydrogen bond.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4490426&req=5

ijms-16-12014-f007: Lanosterol and itraconazole (ITC) binding in CYP51F1. (A) Lanosterol is depicted with carbon atoms colored cyan and heme with carbon atoms colored yellow. Selected oxygen atoms are colored red; nitrogen is blue; and iron is stone blue; (B) ITC is depicted with carbon atoms colored green, and other atoms are colored as in (A). A 2D diagram of the interaction between the protein and ligands is shown below: (a) lanosterol and (b) ITC. Purple dot: amino acids for a hydrogen bond, static electricity and polarity interactions. Green dot: amino acids for VDW (Van der Waals) interactions. Blue shadow: solvent around the amino acids and atoms. Green arrow: electron donor for a hydrogen bond.
Mentions: The cavity volume at the binding sites was calculated, using DS (Binding Site module) for the CYP51F1 protein structure. A binding site with a maximum volume (x: 22.488, y: 8.09, z: 13.729; volume: 836.5) was selected manually, as it should have the appropriate volume for a molecule to adopt minimal energy. For monoterpenes (limonene, 3-carene and pinene) and sesquiterpenes (β-caryophyllene and longifolene), one pose of each was generated using “LibDock.” Seven DS scoring functions (Ligscore1, Ligscore2, -PLP1, -PLP2, Jain, -PMF and -PMF04) and consensus scoring functions were used to re-evaluate the position of docked molecules (Table 4). The position of terpenoid molecules were shown in the binding pocket of CYP51F1 colored green (Figure 6). The interactions of CYP51F1-terpenoid molecules were shown in a 2D diagram (Figure S1). For lanosterol and ITC, the most suitable docking mode for each molecule received a consensus score of seven and six, respectively. More than twelve amino acid residues participated in the interactions between CYP51F1 and the ligands lanosterol and ITC (Figure 7). Three (Ala 302, 306, Leu 303) and six (Ala 306, Met 70, His 373, Gly 69, Ser 374, Hem 601) amino acid residues formed hydrogen bonds, static electricity and polarity interactions between CYP51F1 and the ligands lanosterol and ITC, respectively (Figure 7). These sixteen amino acid residues (Ala 507, Val 135, Phe 130, 229, 234, Met 376, Thr 126, Leu 509, Tyr 68, 122, 136, 506, Pro 231, Ile 372, 375 and Ser 508) formed Van der Waals (VDW) interactions in both CYP51F1-lanosterol and CYP51F1-ITC (Figure S2).

Bottom Line: We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae).The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1) and transmembrane helix 1 (TMH1).The minimal inhibitory concentrations (MIC) of terpenoid and azole fungicides (itraconazole (ITC)) and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.

View Article: PubMed Central - PubMed

Affiliation: College of Forestry, Northwest A&F University, Yangling 712100, China. dailulu@nwafu.edu.cn.

ABSTRACT
Leptographium qinlingensis is a fungal associate of the Chinese white pine beetle (Dendroctonus armandi) and a pathogen of the Chinese white pine (Pinus armandi) that must overcome the terpenoid oleoresin defenses of host trees. L. qinlingensis responds to monoterpene flow with abundant mechanisms that include export and the use of these compounds as a carbon source. As one of the fungal cytochrome P450 proteins (CYPs), which play important roles in general metabolism, CYP51 (lanosterol 14-α demethylase) can catalyze the biosynthesis of ergosterol and is a target for antifungal drug. We have identified an L. qinlingensis CYP51F1 gene, and the phylogenetic analysis shows the highest homology with the 14-α-demethylase sequence from Grosmannia clavigera (a fungal associate of Dendroctonus ponderosae). The transcription level of CYP51F1 following treatment with terpenes and pine phloem extracts was upregulated, while using monoterpenes as the only carbon source led to the downregulation of CYP5F1 expression. The homology modeling structure of CYP51F1 is similar to the structure of the lanosterol 14-α demethylase protein of Saccharomyces cerevisiae YJM789, which has an N-terminal membrane helix 1 (MH1) and transmembrane helix 1 (TMH1). The minimal inhibitory concentrations (MIC) of terpenoid and azole fungicides (itraconazole (ITC)) and the docking of terpenoid molecules, lanosterol and ITC in the protein structure suggested that CYP51F1 may be inhibited by terpenoid molecules by competitive binding with azole fungicides.

No MeSH data available.