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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.


Multiple sequence alignment. The alignment included CYP51F1 from L. qinlingensis, the Saccharomyces cerevisiae YJM789 lanosterol 14-α demethylase protein (PDB code: 4k0f chain A) sequence and the genes used in Figure 1. The heme-binding region (FXXGXRXCXG), PERF domain (PXRX) and K-helix (EXXR) are shown with an underline. Substrate recognition sites (SRSs) 1–2 and 4–6 were manually determined according to 4k0f-A. The predicted membrane interactions of 4k0f-A (MH1 and TMH1) are in grey boxes.
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ijms-16-12014-f002: Multiple sequence alignment. The alignment included CYP51F1 from L. qinlingensis, the Saccharomyces cerevisiae YJM789 lanosterol 14-α demethylase protein (PDB code: 4k0f chain A) sequence and the genes used in Figure 1. The heme-binding region (FXXGXRXCXG), PERF domain (PXRX) and K-helix (EXXR) are shown with an underline. Substrate recognition sites (SRSs) 1–2 and 4–6 were manually determined according to 4k0f-A. The predicted membrane interactions of 4k0f-A (MH1 and TMH1) are in grey boxes.

Mentions: The full-length sequences of CYP51F1 (named by the P450 nomenclature committee) gene was 1996 bp with a 1590-bp open reading frame (ORF), which encoded 529 amino acids (Table 2). The CYP51F1 gene was flanked by 5ʹ and 3ʹ untranslated regions (UTRs) varying in length from 206–200 bp. The predicted molecular mass was 59.31 kDa with an isoelectric point of 6.82 (Table 2). The predicted subcellular localization of the P450 protein revealed a putative code for a typical membrane protein containing approximately 20 hydrophobic residues that are likely membrane anchors in the endoplasmic reticulum (Table 2). The alignment and comparison of the deduced amino acid sequence of CYP51F1 from L. qinlingensis with the S. cerevisiae YJM789 lanosterol 14-α demethylase protein sequence allowed for the identification of the substrate recognition sites (Figure 2).


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)

Multiple sequence alignment. The alignment included CYP51F1 from L. qinlingensis, the Saccharomyces cerevisiae YJM789 lanosterol 14-α demethylase protein (PDB code: 4k0f chain A) sequence and the genes used in Figure 1. The heme-binding region (FXXGXRXCXG), PERF domain (PXRX) and K-helix (EXXR) are shown with an underline. Substrate recognition sites (SRSs) 1–2 and 4–6 were manually determined according to 4k0f-A. The predicted membrane interactions of 4k0f-A (MH1 and TMH1) are in grey boxes.
© Copyright Policy
Related In: Results  -  Collection

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

ijms-16-12014-f002: Multiple sequence alignment. The alignment included CYP51F1 from L. qinlingensis, the Saccharomyces cerevisiae YJM789 lanosterol 14-α demethylase protein (PDB code: 4k0f chain A) sequence and the genes used in Figure 1. The heme-binding region (FXXGXRXCXG), PERF domain (PXRX) and K-helix (EXXR) are shown with an underline. Substrate recognition sites (SRSs) 1–2 and 4–6 were manually determined according to 4k0f-A. The predicted membrane interactions of 4k0f-A (MH1 and TMH1) are in grey boxes.
Mentions: The full-length sequences of CYP51F1 (named by the P450 nomenclature committee) gene was 1996 bp with a 1590-bp open reading frame (ORF), which encoded 529 amino acids (Table 2). The CYP51F1 gene was flanked by 5ʹ and 3ʹ untranslated regions (UTRs) varying in length from 206–200 bp. The predicted molecular mass was 59.31 kDa with an isoelectric point of 6.82 (Table 2). The predicted subcellular localization of the P450 protein revealed a putative code for a typical membrane protein containing approximately 20 hydrophobic residues that are likely membrane anchors in the endoplasmic reticulum (Table 2). The alignment and comparison of the deduced amino acid sequence of CYP51F1 from L. qinlingensis with the S. cerevisiae YJM789 lanosterol 14-α demethylase protein sequence allowed for the identification of the substrate recognition sites (Figure 2).

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.