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

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.

Keywords: Leptographium qinlingensis; homology modeling; lanosterol 14-α demethylase; molecule docking; terpenoid.

Figure 1

A maximum likelihood tree of cytochrome P450 gene from L. qinlingensis with partial sequences was performed using the amino acidic substitution model WAG model with a Gamma (−lnL = 2883.91). The CYP51F1 from L. qinlingensis is shown with an underline. The values of the bootstrap after 500 pseudoreplicates are shown at the nodes.

Figure 2

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.

Figure 3

The quantitative expression of the CYP51 gene (mean ± SE) in mycelia grown on CM (complete medium) + methanol and CW (Chinese white pine phloem methanol extract (CWPPE)) and T (terpenoid blend) of L. qinlingensis. CYP expression was normalized with respect to EF1. The 2^−ΔΔCt and SE values were transformed at log₂ for plotting.

Figure 4

Quantitative expression of the CYP51 gene (mean ± SE) in L. qinlingensis grown on YNB (yeast nitrogen base without amino acids) + Ma (mannose) and MT (monoterpenes) and OA (oleic acid) with different carbon sources. CYP expression was normalized with respect to EF1. The 2^−ΔΔCt and SE values were transformed at log₂ for plotting.

Figure 5

Superimposition of the model protein CYP51F1 (cyan) and template protein 4k0f A (yellow). The superimposed structure of the protein CYP51F1 with the template protein 4k0f chain A demonstrates an RMSD (Root-mean-square deviation) value of 10.805 Å. The predicted membrane interactions of 4k0f chain A (MH1 and TMH1) were added as two slices (cytoplasmic membrane in blue and luminal membrane in green).

Figure 6

The binding pocket of CYP51F1 with terpenoid molecules. The terpenoid molecules limonene (A); carene (B); pinene (C); β-caryophyllene (D) and longifolene (E) are drawn as a green stick representation. The heme-porphyrin is drawn as a ball and stick representation with carbon atoms in cyan.

Figure 7

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.