Cloning And Expression Of CYP51 From Penicillium Digitatum And Application In Screening Novel 14α-demethylation Inhibitors

Green mold of citrus,caused by Penicillium digitatum Sacc,is the most serious postharvest disease of citrus.P.digitatum is the most harmful citrus fruit pathogen and infects the fruit during harvesting and processing.Up to date,14α-demethylation inhibitors(DMIs)are an important group of sterol biosynthesis inhibitors(SBIs)widely used in agriculture.Sterol 14α-demethylase(CYP51)is the only family of P450s,which is widely distributed in different biological kingdoms,found in animals,plants,fungi,yeast,lower eukaryotes and bacteria and considered to be the most ancient member of the superfamily.Cytochrome P450 14α-sterol demethylases are essential enzymes in sterol biosynthesis in eukaryotes.It removes the 14α-methyl group from sterol precursors such as lanosterol,obtusifoliol,dihydrolanosterol,and 24(28)-methylene-24,25-dihydrolanosterol.Inhibitors of CYP51 include triazole antifungal agents,such as fluconazole and itraconazole,drugs used in treatment of topical and systemic mycoses.Sterol biosynthesis is one of the general metabolic pathways present in the majority of eukaryotic cells and organisms.It leads to production of cholesterol in animals,ergosterol in fungi,and a variety of phytosterols in plants.The biosynthesis of sterol will lead to the damage or loss of structure and function of membrane via the inhibitation of 14α-demethylase,which will lead to the death of eukaryotes at last.In this study,sterol 14α-demethylase gene from P.digitatum was cloned and expressed,and the expressed product of the gene and the preliminary function were analyzed.Meanwhile,the 3D model was built and the fungicidal activities of some chemical entitles were screened according to the spectrophotometric method.The results are listed as follows:1.The P.digitatum CYP51 gene(PdCYP51)was cloned.Compared with the DNA sequence of P. digitatum strain PD5(accession no.AB030178),there were four nucleotide changed(C1370→T, A1604→G,G1648→A and G1768→T)resulting in four different amino acids(P345L,E423G, G438R,V478L).The cDNA sequence was registered in Genbank(accession no.DQ355161). When compared to other amino acid sequences of CYP51 in Genbank,it indicated that PdCYP51 (ABC87815,Pd1)had 99%identities to that of P.digitatum strain PD5(BAB03658,Pd2),86%to P.italicum(Q12664,Pi),67%to Aspergillus fumigatus(AAF32372,Af)and Neosartorya fischeri (XP₀01267338,Nf).All of the sequences contains SRS1(YxxF/LxxPxFGxxVxF/YD/a),SRS4 (GQ/Hht/sS)and heine-binding protein sequence(FxxGxxxCxG)and four mutated amino acids all located outside the conserved regions.The four mutated amino acids have little relation with drug resistance according to the bioassay. 2.The three-dimensional(3D)model of PdCYP51 was built based on the structure template of le9x.pdb(Mycobacterium tuberculosis,MTCYP51)using FUGUE module of SYBYL7.0 program.The conserved amino acids of the activity cave were superimposed with the template. CYP51 is a kind of trans-membrane protein,displaying key roll in the biosynthesis of sterol. According to the analysis of hydrophobicity,signal peptide and transmembrane domain of PdCYP51,the N-terminus has 66 amino acids which stride across membrane twice,and the hydrophobic domain is mainly located in the transmembrane domain and the catalytic cave of the protein.The recombinant plasmid pET-PdCYP51 was constructed,and the 59kDa protein (PdCYP51)was obtained when induced by IPTG in E.coli BL21(DE3)strain at 37℃. Antibodies against purified PdCYP51 recombinant protein were raised in rabbit and western blotting was performed.It was indicated that the purified PdCYP51 had immunologic activity.3.The expressions of PdCYP51 were optimized under various conditions,and the soluble recombinant protein PdCYP51 was obtained in E.coli BL21(DE3)Rosetta at 25-30℃at last. According to the analysis of the transmemebrane structure and the homology modeling,various mutanted recombinant plasmids of pET-PdCYP51-18,pET-PdCYP51-66,pET-PdCYP51-244, p6P-PdCYP51-244 and p9K-PdCYP51-244.PdCYP51 were constructed and expressed in E.coli BL21(DE3).The recombinant eukaryotic expression vector of pAUR-PdCYP51,p9K-PdCYP51 and p9K-PdCYP51-244 were also constructed.4.The activity of the soluble recombinant protein PdCYP51 was detected according to the CO difference spectra.The binding effect of the PdCYP51 with the representable antifungal agents, such as diniconazole,tebuconazole,triadimenol and triadimefon,were investigated through the binding spectra under various enzyme activity,enzyme purity and concentration.When compared with bioassay results,the binding spectra showed that the activity and proper concentration of the enzyme were necessary for obtaining accurate binding spectra.According to Michaelis-Menten equation,The Kd values of diniconazole,tebuconazole,triadimenol and triadimefon were 0.12μM,0.32μM,0.64μM and 0.99μM respectively,which significantly correlated to their EC₅₀ values on the growth of P.digitatum.These results indicated that the binding spectra of fungicide with sterol 14α-demethylase can serve as a reliable and quick method for the screening of novel fungicides.5.The activity of heterogenetic expressed PdCYP51 were investigated to build a binding-spectra method in screening the fungicidal activities of novel chemical entitles(serials XF,serials WJ and serials ZST).According to the binding-spectra data and the bioassay results,some novel compounds with high activity were found,such as XF-22,XF-50,XF-78,XF-100,XF-113, XF118 and XF169 of serials XF,and WJ7,WJ10,WJ11,WJ12,WJ15,WJ21,WJ22,WJ25 and WJ26 of serials WJ and ZST1,ZST5 and ZST9 of serials ZST.The Kd of WJ7,WJ10,WJ11,WJ12,WJ15,WJ21,WJ22,WJ25 and WJ26 were all less than 0.2μM,and the inhibition ratio all arrived at 100%on the growth of P.digitatum.Some chemical compounds even arrived at 80%at the concentration of 25mg/ml.6.The novel chemical entitles were docked to heme atom of the catalysis core of PdCYP51 by FLexX.In the binding model,the distance between N atom of chemical entitles and heme atom of the catalysis core of PdCYP51 was 3(?)obtained by computer aided design(CAD).The typeâ…¡difference spectral results also confirmed the reliability of the homology model,and showed that the selection criteria for the binding model of the compound were reliable,which provide an effective strategy to design novel compounds with high affectivity and specificity.