Heterologous Expression and Homology Modelling of Sterol 14α-demethylase of Fusarium graminearum and Its Interaction with azoles
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- 英文论文题名：Heterologous Expression and Homology Modelling of Sterol 14α-demethylase of Fusarium graminearum and Its Interaction with azoles
- 论文作者：QIAN Heng-wei ; CHI Meng-yu ; ZHAO Ying ; HUANG Jin-guang
- 英文论文作者：QIAN Heng-wei ; CHI Meng-yu ; ZHAO Ying ; HUANG Jin-guang ; College of Agronomy and Plant Protection ; QingDao Agriculture University ; Key Lab of Integrated Crop Disease and Pest Management of Shandong Province
- 年：2016
- 作者机构：College of Agronomy and Plant Protection,QingDao Agriculture University;Key Lab of Integrated Crop Disease and Pest Management of Shandong Province;
- 英文论文关键词：Fusarium graminearum ; CYP51 ; Expression ; Homology modeling ; Molecular docking
- 会议召开时间：2016-08-05
- 会议录名称：中国植物病理学会2016年学术年会论文集
- 英文会议录名称：Proceedings of the Annual Meeting of Chinese Society for Plant Pathology(2016)
- 语种：英文
- 分类号：S435.121.45
- 学会代码：ZGVS
- 会议名称：中国植物病理学会2016年学术年会
- 会议地点：中国江苏南京
- 主办单位：中国植物病理学会
- 学会名称：中国植物病理学会
- 页数：2
- 文件大小：86k
- 原文格式：O
- 会议级别：全国
摘要
Fusarium graminearum is the main pathogen of Fusarium Head Blight(FHB),a worldwide plant disease and one of the major wheat diseases in China.The infected wheat grain could product the mycotoxins and posed a serious threat to human and animal health.The control of the FHB is main dependent on the application of fungicides,Demethylase inhibitors(DMI fungicides,DMIs),such as tebuconazole and diniconazole,which inhibit the biosynthesis of ergosterol in fungi because of sterol 14α-demethylase(CYP51) binding.Ergosterol is essential for cell membrane fluidity and permeability,so ergosterol depletion will result in the inhibition of fungal growth.Due to the intensive use of DMI fungicides in agriculture,resistance has been detected in infected crops and become more serious in recent years.F.graminearum has three CYP51 genes,CYP51 A,CYP51B and CYP51 C,which share 61.65%amino acid sequence identity.CYP51 B is expressed constitutively,while gene CYP51 A is reported to be inducible.Fg CYP51 B,as the most conserved CYP51 gene in all fungi,encodes the enzyme primarily responsible for sterol 14α-demethylation.In order to explore the molecular mechanism of drug resistance of CYP51,we amplified the FgCYP51B(FGSG_01000) fragments by PCR and cloned into Escherichia coli expression vector pETM-30,the constructed plasmids were transformed into E.coli strain BL21(DE3).After IPTG induced,it was not expressed in E.coli verified by SDS-PAGE.According to TMHMM Server V.2.0 prediction,the N-terminal 49 aa(transmembrane domains) of FgCYP51 B was truncated(T-FgCYP51B).T-FgCYP51 B was successfully expressed in E.coli after IPTG induced,but the protein mainly existed in the form of inclusion body after affinity chromatography.Fungal CYP51 proteins are membrane bound,making it more difficult to study their structural compared with the solube bacterial CYP51.So homology modeling and molecular docking with ligand has been used extensively to explain possible protein-drug interactions.The FgCYP51 B structure was modeled based on the crystal structure of AfCYP51B(PDB ID:4UYM) as the template using the Swiss-Model server,then energy minimization was performed to eliminate improper contacts for the modeled structure in Amber.The structures of five small molecular inhibitors,including Diniconazole(DIC),Tebuconazole(TEC),Triadimenol(TRL),Triadimefon(TRN) and Propiconazole(PRC),were conducted geometry optimization by Gaussian programe.AutoDock vina was then used to dock the inhibitors into the active cavity of the enzyme respectively.The structure of complexes were visualized using PyMOL.Modeled structure exhibiting a high degree of sequence identity with template is in consistant with the view and outlines a highly hydrophobic cavity consisting of a number of hydrophobic residues,mainly including Tyr123,Phe131,Tyr137,Phe230,Leu510 and Phe511,which are conserved.Tyr123,Phe131 and Tyr137 of FgCYP51 are highly conserved across all biological kingdoms,while Phe230 is conserved across fungal species,Leu510 and Phe511 varies in different phyla.Thus,the non-conservative and Phe230,Leu510 and Phe511 of FgCYP51 are supposed to be key residues interacting with azole fungicides.Elucidation of molecular mechanisms of protein-drug interactions is underway.In summary,our results offer new insights into resistance molecular mechanisms of F.graminearum.
Fusarium graminearum is the main pathogen of Fusarium Head Blight(FHB),a worldwide plant disease and one of the major wheat diseases in China.The infected wheat grain could product the mycotoxins and posed a serious threat to human and animal health.The control of the FHB is main dependent on the application of fungicides,Demethylase inhibitors(DMI fungicides,DMIs),such as tebuconazole and diniconazole,which inhibit the biosynthesis of ergosterol in fungi because of sterol 14α-demethylase(CYP51) binding.Ergosterol is essential for cell membrane fluidity and permeability,so ergosterol depletion will result in the inhibition of fungal growth.Due to the intensive use of DMI fungicides in agriculture,resistance has been detected in infected crops and become more serious in recent years.F.graminearum has three CYP51 genes,CYP51 A,CYP51B and CYP51 C,which share 61.65%amino acid sequence identity.CYP51 B is expressed constitutively,while gene CYP51 A is reported to be inducible.Fg CYP51 B,as the most conserved CYP51 gene in all fungi,encodes the enzyme primarily responsible for sterol 14α-demethylation.In order to explore the molecular mechanism of drug resistance of CYP51,we amplified the FgCYP51B(FGSG_01000) fragments by PCR and cloned into Escherichia coli expression vector pETM-30,the constructed plasmids were transformed into E.coli strain BL21(DE3).After IPTG induced,it was not expressed in E.coli verified by SDS-PAGE.According to TMHMM Server V.2.0 prediction,the N-terminal 49 aa(transmembrane domains) of FgCYP51 B was truncated(T-FgCYP51B).T-FgCYP51 B was successfully expressed in E.coli after IPTG induced,but the protein mainly existed in the form of inclusion body after affinity chromatography.Fungal CYP51 proteins are membrane bound,making it more difficult to study their structural compared with the solube bacterial CYP51.So homology modeling and molecular docking with ligand has been used extensively to explain possible protein-drug interactions.The FgCYP51 B structure was modeled based on the crystal structure of AfCYP51B(PDB ID:4UYM) as the template using the Swiss-Model server,then energy minimization was performed to eliminate improper contacts for the modeled structure in Amber.The structures of five small molecular inhibitors,including Diniconazole(DIC),Tebuconazole(TEC),Triadimenol(TRL),Triadimefon(TRN) and Propiconazole(PRC),were conducted geometry optimization by Gaussian programe.AutoDock vina was then used to dock the inhibitors into the active cavity of the enzyme respectively.The structure of complexes were visualized using PyMOL.Modeled structure exhibiting a high degree of sequence identity with template is in consistant with the view and outlines a highly hydrophobic cavity consisting of a number of hydrophobic residues,mainly including Tyr123,Phe131,Tyr137,Phe230,Leu510 and Phe511,which are conserved.Tyr123,Phe131 and Tyr137 of FgCYP51 are highly conserved across all biological kingdoms,while Phe230 is conserved across fungal species,Leu510 and Phe511 varies in different phyla.Thus,the non-conservative and Phe230,Leu510 and Phe511 of FgCYP51 are supposed to be key residues interacting with azole fungicides.Elucidation of molecular mechanisms of protein-drug interactions is underway.In summary,our results offer new insights into resistance molecular mechanisms of F.graminearum.
Fusarium graminearum is the main pathogen of Fusarium Head Blight(FHB),a worldwide plant disease and one of the major wheat diseases in China.The infected wheat grain could product the mycotoxins and posed a serious threat to human and animal health.The control of the FHB is main dependent on the application of fungicides,Demethylase inhibitors(DMI fungicides,DMIs),such as tebuconazole and diniconazole,which inhibit the biosynthesis of ergosterol in fungi because of sterol 14α-demethylase(CYP51) binding.Ergosterol is essential for cell membrane fluidity and permeability,so ergosterol depletion will result in the inhibition of fungal growth.Due to the intensive use of DMI fungicides in agriculture,resistance has been detected in infected crops and become more serious in recent years.F.graminearum has three CYP51 genes,CYP51 A,CYP51B and CYP51 C,which share 61.65%amino acid sequence identity.CYP51 B is expressed constitutively,while gene CYP51 A is reported to be inducible.Fg CYP51 B,as the most conserved CYP51 gene in all fungi,encodes the enzyme primarily responsible for sterol 14α-demethylation.In order to explore the molecular mechanism of drug resistance of CYP51,we amplified the FgCYP51B(FGSG_01000) fragments by PCR and cloned into Escherichia coli expression vector pETM-30,the constructed plasmids were transformed into E.coli strain BL21(DE3).After IPTG induced,it was not expressed in E.coli verified by SDS-PAGE.According to TMHMM Server V.2.0 prediction,the N-terminal 49 aa(transmembrane domains) of FgCYP51 B was truncated(T-FgCYP51B).T-FgCYP51 B was successfully expressed in E.coli after IPTG induced,but the protein mainly existed in the form of inclusion body after affinity chromatography.Fungal CYP51 proteins are membrane bound,making it more difficult to study their structural compared with the solube bacterial CYP51.So homology modeling and molecular docking with ligand has been used extensively to explain possible protein-drug interactions.The FgCYP51 B structure was modeled based on the crystal structure of AfCYP51B(PDB ID:4UYM) as the template using the Swiss-Model server,then energy minimization was performed to eliminate improper contacts for the modeled structure in Amber.The structures of five small molecular inhibitors,including Diniconazole(DIC),Tebuconazole(TEC),Triadimenol(TRL),Triadimefon(TRN) and Propiconazole(PRC),were conducted geometry optimization by Gaussian programe.AutoDock vina was then used to dock the inhibitors into the active cavity of the enzyme respectively.The structure of complexes were visualized using PyMOL.Modeled structure exhibiting a high degree of sequence identity with template is in consistant with the view and outlines a highly hydrophobic cavity consisting of a number of hydrophobic residues,mainly including Tyr123,Phe131,Tyr137,Phe230,Leu510 and Phe511,which are conserved.Tyr123,Phe131 and Tyr137 of FgCYP51 are highly conserved across all biological kingdoms,while Phe230 is conserved across fungal species,Leu510 and Phe511 varies in different phyla.Thus,the non-conservative and Phe230,Leu510 and Phe511 of FgCYP51 are supposed to be key residues interacting with azole fungicides.Elucidation of molecular mechanisms of protein-drug interactions is underway.In summary,our results offer new insights into resistance molecular mechanisms of F.graminearum.
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