反枝苋乙酰乳酸合成酶与烟嘧磺隆分子结合模式分析及抗性位点预测
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摘要
为研究反枝苋对乙酰乳酸合成酶(ALS)抑制剂的抗性机制,本研究根据反枝苋的ALS氨基酸序列,利用同源模建的方法构建了其三维结构,并采用分子对接和分子动力学模拟的方法预测了反枝苋ALS与烟嘧磺隆分子的结合模式。根据结合模式对已报道的Pro 197和Trp 574等位点突变产生抗性的原因进行了分析。结果发现:Pro 197和Trp 574等位点的残基与烟嘧磺隆分子之间存在重要的疏水作用和π-π作用等其他相互作用,或该位点的残基具有特殊结构影响着通道形状。分析表明,ALS与烟嘧磺隆之间的氢键、疏水作用等非共价相互作用以及通道形状的改变都有可能影响二者结合稳定性,从而使杂草产生抗性。基于此结论,本研究预测Val 196、Met 200、Phe 206和Lys 256突变同样可能使杂草对ALS抑制剂敏感度发生变化。本研究利用计算机模拟技术分析了ALS抗性机制,为反抗性除草剂的分子设计提供了指导。
In order to study the resistance mechanism of Amaranthus retroflexus L. to acetolactate synthase(ALS) inhibitor, homologous modeling was conducted to construct the three-dimensional structure of ALS based on its amino acid sequence of Amaranthus retroflexus L.. Molecular docking and molecular dynamics simulations were used to predict the binding mode between the ALS of Amaranthus retroflexus L. and nicosulfuron. Based on the binding mode, analysis of the resistance reason at the reported mutations of Pro 197, Trp 574 and other sites indicated the existence of important hydrophobic interactions, π-π interactions and other interactions between the residues of the above site and nicosulfuron, or special structures of the residues that affected the channel shape. Analysis shows that non-covalent interactions between the ALS and nicosulfuron, such as hydrogen bonding and hydrophobic interactions, and changes in the channels shape might affect the binding stability and cause the weeds resistance. These results predicted that the mutations at Val 196, Met 200, Phe 206 and Lys256 might also change the sensitivity of weeds to ALS inhibitors. In this study, the ALS resistance mechanism was analyzed using computer simulation techniques, and the results provided a reference for the design of anti-resistant herbicides.
In order to study the resistance mechanism of Amaranthus retroflexus L. to acetolactate synthase(ALS) inhibitor, homologous modeling was conducted to construct the three-dimensional structure of ALS based on its amino acid sequence of Amaranthus retroflexus L.. Molecular docking and molecular dynamics simulations were used to predict the binding mode between the ALS of Amaranthus retroflexus L. and nicosulfuron. Based on the binding mode, analysis of the resistance reason at the reported mutations of Pro 197, Trp 574 and other sites indicated the existence of important hydrophobic interactions, π-π interactions and other interactions between the residues of the above site and nicosulfuron, or special structures of the residues that affected the channel shape. Analysis shows that non-covalent interactions between the ALS and nicosulfuron, such as hydrogen bonding and hydrophobic interactions, and changes in the channels shape might affect the binding stability and cause the weeds resistance. These results predicted that the mutations at Val 196, Met 200, Phe 206 and Lys256 might also change the sensitivity of weeds to ALS inhibitors. In this study, the ALS resistance mechanism was analyzed using computer simulation techniques, and the results provided a reference for the design of anti-resistant herbicides.
引文
[1]GARCIA M D,WANG J G,LONHIENNE T,et al.Crystal structure of plant acetohydroxyacid synthase,the target for several commercial herbicides[J].FEBS J,2017,284(13):2037-2051.
[2]陈伟,魏巍,周莎,等.新型含苯基取代嘧啶基磺酰脲衍生物的设计、合成及生物活性[J].高等学校化学学报,2015,36(4):672-681.CHEN W,WEI W,ZHOU S,et al.Design,synthesis and biological activity of novel sulfonylurea derivatives containing phenylsubstituted pyrimidine moiety[J].Chem J Chinese Univ,2015,36(4):672-681.
[3]王建国.乙酰乳酸合成酶及其抑制剂研究新进展[J].农药学学报,2014,16(4):367-374.WANG J G.Recent progress on acetohydroxyacid synthase and its inhibitors[J].Chin J Pestic Sci,2014,16(4):367-374.
[4]汪冒君,宣南霞,吴军.乙酰乳酸合成酶与抑制剂ZJ0777及CIE的分子对接和分子动力学模拟[J].浙江大学学报(理学版),2015,42(6):709-713.WANG M J,XUAN N X,WU J.Molecular docking and molecular dynamic simulations of inhibitor ZJ0777 and CIE binding to acetohydroxyacid synthases[J].J Zhejiang Univ(Sci Ed),2015,42(6):709-713.
[5]NAKKA S,THOMPSON C R,PETERSON D E,et al.Target sitebased and non-target site based resistance to ALS inhibitors in palmer amaranth(Amaranthus palmeri)[J].Weed Sci,2017,65(6):681-689.
[6]RIAR D S,TEHRANCHIAN P,NORSWORTHY J K,et al.Acetolactate synthase-inhibiting,herbicide-resistant rice flatsedge(Cyperus iria):cross-resistance and molecular mechanism of resistance[J].Weed Sci,2017,63(4):748-757.
[7]QIN Z,ZHANG J E,JIANG Y P,et al.Invasion process and potential spread of Amaranthus retroflexus in China[J].Weed Res,2018,58(1):57-67.
[8]邓维.抗苯磺隆播娘蒿抗性机理及抗性突变对乙酰乳酸合成酶功能影响[D].中国农业大学,2017.DENG W.The resistance mechanisms of tribenuron-methyl-resistant flixweed(Descurainia sophia L.)and effects of resistance-endowing mutations on ALS functionality[D].Beijing:China Agricultural University,2017.
[9]SIBONY M,MICHEL A,HAAS H U,et al.Sulfometuron-resistant Amaranthus retroflexus:cross-resistance and molecular basis for resistance to acetolactate synthase-inhibiting herbicides[J].Weed Res,2001,41(6):509-522.
[10]MCNAUGHTON K E,LETARTE J,LEE E A,et al.Mutations in ALS confer herbicide resistance in redroot pigweed(Amaranthus retroflexus)and Powell amaranth(Amaranthus powellii)[J].Weed Sci,2005,53(1):17-22.
[11]李兰芬,南洁,苏晓东.蛋白质晶体学技术的发展与展望[J].生物物理学报,2007,23(4):246-255.LI L F,NAN J,SU X D.Protein crystallography technology:past,present and future[J].Acta Biophysica Sinica,2007,23(4):246-255.
[12]PANG S S,GUDDAT L W,DUGGLEBY R G.Crystallization of the catalytic subunit of Saccharomyces cerevisiae acetohydroxyacid synthase[J].Acta Crystallographica Section D:BiologicalCrystallography,2001,57(9):1321-1323.
[13]刘吉元.蛋白质与配体相互作用分子模拟研究[D].杨凌:西北农林科技大学,2014.LIU J Y.In silico study of protein-ligand interactions[D].Yangling:Northwest A&F University,2014.
[14]CLARK A J,TIWARY P,BORRELLI K,et al.Prediction of proteinligand binding poses via a combination of induced fit docking and metadynamics simulations[J].J Chem Theory Comput,2016,12(6):2990-2998.
[15]LASKOWSKI R A,MACARTHUR M W,MOSS D S,et al.PROCHECK:a program to check the stereochemical quality of protein structures[J].J Appl Crystallogr,1993,26(2):283-291.
[16]COLOVOS C,YEATES T O.Verification of protein structures:patterns of nonbonded atomic interactions[J].Protein Sci,1993,2(9):1511-1519.
[17]EISENBERG D,LüTHY R,BOWIE J U.[20]VERIFY3D:Assessment of protein models with three-dimensional profiles[J].Methods Enzymol,1997,277:396-404.
[18]赵斌,霍静倩,邢继红,等.拟南芥转酮醇酶蛋白AtTKL1的同源建模及与α-三联噻吩结合作用分析[J].高等学校化学学报,2015,36(4):682-686.ZHAO B,HUO J Q,XING J H,et al.Homologous modeling of transketolase at TKL1 and its combination withα-terthienyl in Arabidopsis thaliana[J].Chem J Chinese Univ,2015,36(4):682-686.
[19]LONHIENNE T,GARCIA M D,PIERENS G,et al.Structural insights into the mechanism of inhibition of AHAS by herbicides[J].Proc Natl Acad Sci USA,2018,115(9):E1945-E1954.
[20]GARCIA M D,NOUWENS A,LONHIENNE T G,et al.Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families[J].Proc Natl Acad Sci USA,2017,114(7):E1091-E1100.
[21]WANG J G,LEE P K M,DONG Y H,et al.Crystal structures of two novel sulfonylurea herbicides in complex with Arabidopsis thaliana acetohydroxyacid synthase[J].FEBS J,2009,276(5):1282-1290.
[22]LONHIENNE T,NOUWENS A,WILLIAMS C M,et al.Commercial herbicides can trigger the oxidative inactivation of acetohydroxyacid synthase[J].Angew Chem,2016,55(13):4247-4251.
[23]DUGGLEBY R G,MCCOURT J A,GUDDAT L W.Structure and mechanism of inhibition of plant acetohydroxyacid synthase[J].Plant Physiol Biochem,2008,46(3):309-324.
[24]CHIPMAN D,BARAK Z,SCHLOSS J V.Biosynthesis of 2-aceto-2-hydroxy acids:acetolactate synthases and acetohydroxyacid synthases[J].Biochimica et Biophysica Acta(BBA)-Protein Structure and Molecular Enzymology,1998,1385(2):401-419.
[25]XIONG Y,LIU J J,YANG G F,et al.Computational determination of fundamental pathway and activation barriers for acetohydroxyacid synthase-catalyzed condensation reactions of ofα-keto acids[J].JComput Chem,2010,31(8):1592-1602.
[26]YU Q,POWLES S B.Resistance to AHAS inhibitor herbicides:current understanding[J].Pest Manag Sci,2014,70(9):1340-1350.
[27]张敏,荆晶晶,李成涛,等.结合分子模拟探讨不同醚键PBS基共聚物的性能与酶催化降解[J].高等学校化学学报,2014,35(12):2706-2712.ZHANG M,JING J J,LI C T,et al.Performance of PBS-based copolymer containing different ether bond and its enzymatic degradation by molecular simulation[J].Chem J Chinese Univ,2014,35(12):2706-2712.
[28]吕婧,蒋勇军,俞庆森,等.洋刀豆脲酶与抑制剂相互作用的分子对接和分子动力学研究[J].化学学报,2011,69(20):2427-2433.LV J,JIANG Y J,YU Q S,et al.Molecular docking and molecular dynamics simulations of inhibitors binding to jack bean urease[J].Acta Chimica Sinica,2011,69(20):2427-2433.
[29]KUMAR V,JHA P,GIACOMINI D,et al.Molecular basis of evolved resistance to glyphosate and acetolactate synthase-Inhibitor herbicides in Kochia(Kochia scoparia)accessions from montana[J].Weed Sci,2017,63(4):758-769.
[30]MCCOURT J A,PANG S S,KINGSCOTT J,et al.Herbicidebinding sites revealed in the structure of plant acetohydroxyacid synthase[J].Proc Natl Acad Sci USA,2006,103(3):569-573.
[31]NTOANIDOU S,KALOUMENOS N,DIAMANTIDIS G,et al.Molecular basis of Cyperus difformis cross-resistance to ALS-inhibiting herbicides[J].Pestic Biochem Physiol,2016,127:38-45.
[32]王冬梅,吕淑霞.生物化学[M].北京:科学出版社,2010.WANG D M,LV S X.Biochemistry[M].Beijing:Science Press,2010.
[33]POWLES S B,YU Q.Evolution in action:plants resistant to herbicides[J].Annu Rev Plant Biol,2010,61:317-347.
[34]ASHIGH J,CORBETT C A L,SMITH P J,et al.Characterization and diagnostic tests of resistance to acetohydroxyacid synthase inhibitors due to an Asp376 Glu substitution in Amaranthus powellii[J].Pestic Biochem Physiol,2009,95(1):38-46.
[35]CHEN J,HUANG Z,ZHANG C,et al.Molecular basis of resistance to imazethapyr in redroot pigweed(Amaranthus retroflexus L.)populations from China[J].Pestic Biochem Physiol,2015,124:43-47.
[36]WERLE R,BEGCY K,YERKA M K,et al.Independent evolution of acetolactate synthase-inhibiting herbicide resistance in weedy Sorghum populations across common geographic regions[J].Weed Sci,2017,65(1):164-176.
[37]HILL C M,DUGGLEBY R G.Mutagenesis of Escherichia coli acetohydroxyacid synthase isoenzymeⅡand characterization of three herbicide-insensitive forms[J].Biochem J,1998,335(3):653-661.
[38]DUGGLEBY R G,PANG S S,YU H Q,et al.Systematic characterization of mutations in yeast acetohydroxyacid synthase[J].FEBS J,2003,270(13):2895-2904.
[39]JUNG S M,LE D T,YOON S S,et al.Amino acids conferring herbicide resistance in tobacco acetohydroxyacid synthase[J].Bio J,2014,383:53-61.
[2]陈伟,魏巍,周莎,等.新型含苯基取代嘧啶基磺酰脲衍生物的设计、合成及生物活性[J].高等学校化学学报,2015,36(4):672-681.CHEN W,WEI W,ZHOU S,et al.Design,synthesis and biological activity of novel sulfonylurea derivatives containing phenylsubstituted pyrimidine moiety[J].Chem J Chinese Univ,2015,36(4):672-681.
[3]王建国.乙酰乳酸合成酶及其抑制剂研究新进展[J].农药学学报,2014,16(4):367-374.WANG J G.Recent progress on acetohydroxyacid synthase and its inhibitors[J].Chin J Pestic Sci,2014,16(4):367-374.
[4]汪冒君,宣南霞,吴军.乙酰乳酸合成酶与抑制剂ZJ0777及CIE的分子对接和分子动力学模拟[J].浙江大学学报(理学版),2015,42(6):709-713.WANG M J,XUAN N X,WU J.Molecular docking and molecular dynamic simulations of inhibitor ZJ0777 and CIE binding to acetohydroxyacid synthases[J].J Zhejiang Univ(Sci Ed),2015,42(6):709-713.
[5]NAKKA S,THOMPSON C R,PETERSON D E,et al.Target sitebased and non-target site based resistance to ALS inhibitors in palmer amaranth(Amaranthus palmeri)[J].Weed Sci,2017,65(6):681-689.
[6]RIAR D S,TEHRANCHIAN P,NORSWORTHY J K,et al.Acetolactate synthase-inhibiting,herbicide-resistant rice flatsedge(Cyperus iria):cross-resistance and molecular mechanism of resistance[J].Weed Sci,2017,63(4):748-757.
[7]QIN Z,ZHANG J E,JIANG Y P,et al.Invasion process and potential spread of Amaranthus retroflexus in China[J].Weed Res,2018,58(1):57-67.
[8]邓维.抗苯磺隆播娘蒿抗性机理及抗性突变对乙酰乳酸合成酶功能影响[D].中国农业大学,2017.DENG W.The resistance mechanisms of tribenuron-methyl-resistant flixweed(Descurainia sophia L.)and effects of resistance-endowing mutations on ALS functionality[D].Beijing:China Agricultural University,2017.
[9]SIBONY M,MICHEL A,HAAS H U,et al.Sulfometuron-resistant Amaranthus retroflexus:cross-resistance and molecular basis for resistance to acetolactate synthase-inhibiting herbicides[J].Weed Res,2001,41(6):509-522.
[10]MCNAUGHTON K E,LETARTE J,LEE E A,et al.Mutations in ALS confer herbicide resistance in redroot pigweed(Amaranthus retroflexus)and Powell amaranth(Amaranthus powellii)[J].Weed Sci,2005,53(1):17-22.
[11]李兰芬,南洁,苏晓东.蛋白质晶体学技术的发展与展望[J].生物物理学报,2007,23(4):246-255.LI L F,NAN J,SU X D.Protein crystallography technology:past,present and future[J].Acta Biophysica Sinica,2007,23(4):246-255.
[12]PANG S S,GUDDAT L W,DUGGLEBY R G.Crystallization of the catalytic subunit of Saccharomyces cerevisiae acetohydroxyacid synthase[J].Acta Crystallographica Section D:BiologicalCrystallography,2001,57(9):1321-1323.
[13]刘吉元.蛋白质与配体相互作用分子模拟研究[D].杨凌:西北农林科技大学,2014.LIU J Y.In silico study of protein-ligand interactions[D].Yangling:Northwest A&F University,2014.
[14]CLARK A J,TIWARY P,BORRELLI K,et al.Prediction of proteinligand binding poses via a combination of induced fit docking and metadynamics simulations[J].J Chem Theory Comput,2016,12(6):2990-2998.
[15]LASKOWSKI R A,MACARTHUR M W,MOSS D S,et al.PROCHECK:a program to check the stereochemical quality of protein structures[J].J Appl Crystallogr,1993,26(2):283-291.
[16]COLOVOS C,YEATES T O.Verification of protein structures:patterns of nonbonded atomic interactions[J].Protein Sci,1993,2(9):1511-1519.
[17]EISENBERG D,LüTHY R,BOWIE J U.[20]VERIFY3D:Assessment of protein models with three-dimensional profiles[J].Methods Enzymol,1997,277:396-404.
[18]赵斌,霍静倩,邢继红,等.拟南芥转酮醇酶蛋白AtTKL1的同源建模及与α-三联噻吩结合作用分析[J].高等学校化学学报,2015,36(4):682-686.ZHAO B,HUO J Q,XING J H,et al.Homologous modeling of transketolase at TKL1 and its combination withα-terthienyl in Arabidopsis thaliana[J].Chem J Chinese Univ,2015,36(4):682-686.
[19]LONHIENNE T,GARCIA M D,PIERENS G,et al.Structural insights into the mechanism of inhibition of AHAS by herbicides[J].Proc Natl Acad Sci USA,2018,115(9):E1945-E1954.
[20]GARCIA M D,NOUWENS A,LONHIENNE T G,et al.Comprehensive understanding of acetohydroxyacid synthase inhibition by different herbicide families[J].Proc Natl Acad Sci USA,2017,114(7):E1091-E1100.
[21]WANG J G,LEE P K M,DONG Y H,et al.Crystal structures of two novel sulfonylurea herbicides in complex with Arabidopsis thaliana acetohydroxyacid synthase[J].FEBS J,2009,276(5):1282-1290.
[22]LONHIENNE T,NOUWENS A,WILLIAMS C M,et al.Commercial herbicides can trigger the oxidative inactivation of acetohydroxyacid synthase[J].Angew Chem,2016,55(13):4247-4251.
[23]DUGGLEBY R G,MCCOURT J A,GUDDAT L W.Structure and mechanism of inhibition of plant acetohydroxyacid synthase[J].Plant Physiol Biochem,2008,46(3):309-324.
[24]CHIPMAN D,BARAK Z,SCHLOSS J V.Biosynthesis of 2-aceto-2-hydroxy acids:acetolactate synthases and acetohydroxyacid synthases[J].Biochimica et Biophysica Acta(BBA)-Protein Structure and Molecular Enzymology,1998,1385(2):401-419.
[25]XIONG Y,LIU J J,YANG G F,et al.Computational determination of fundamental pathway and activation barriers for acetohydroxyacid synthase-catalyzed condensation reactions of ofα-keto acids[J].JComput Chem,2010,31(8):1592-1602.
[26]YU Q,POWLES S B.Resistance to AHAS inhibitor herbicides:current understanding[J].Pest Manag Sci,2014,70(9):1340-1350.
[27]张敏,荆晶晶,李成涛,等.结合分子模拟探讨不同醚键PBS基共聚物的性能与酶催化降解[J].高等学校化学学报,2014,35(12):2706-2712.ZHANG M,JING J J,LI C T,et al.Performance of PBS-based copolymer containing different ether bond and its enzymatic degradation by molecular simulation[J].Chem J Chinese Univ,2014,35(12):2706-2712.
[28]吕婧,蒋勇军,俞庆森,等.洋刀豆脲酶与抑制剂相互作用的分子对接和分子动力学研究[J].化学学报,2011,69(20):2427-2433.LV J,JIANG Y J,YU Q S,et al.Molecular docking and molecular dynamics simulations of inhibitors binding to jack bean urease[J].Acta Chimica Sinica,2011,69(20):2427-2433.
[29]KUMAR V,JHA P,GIACOMINI D,et al.Molecular basis of evolved resistance to glyphosate and acetolactate synthase-Inhibitor herbicides in Kochia(Kochia scoparia)accessions from montana[J].Weed Sci,2017,63(4):758-769.
[30]MCCOURT J A,PANG S S,KINGSCOTT J,et al.Herbicidebinding sites revealed in the structure of plant acetohydroxyacid synthase[J].Proc Natl Acad Sci USA,2006,103(3):569-573.
[31]NTOANIDOU S,KALOUMENOS N,DIAMANTIDIS G,et al.Molecular basis of Cyperus difformis cross-resistance to ALS-inhibiting herbicides[J].Pestic Biochem Physiol,2016,127:38-45.
[32]王冬梅,吕淑霞.生物化学[M].北京:科学出版社,2010.WANG D M,LV S X.Biochemistry[M].Beijing:Science Press,2010.
[33]POWLES S B,YU Q.Evolution in action:plants resistant to herbicides[J].Annu Rev Plant Biol,2010,61:317-347.
[34]ASHIGH J,CORBETT C A L,SMITH P J,et al.Characterization and diagnostic tests of resistance to acetohydroxyacid synthase inhibitors due to an Asp376 Glu substitution in Amaranthus powellii[J].Pestic Biochem Physiol,2009,95(1):38-46.
[35]CHEN J,HUANG Z,ZHANG C,et al.Molecular basis of resistance to imazethapyr in redroot pigweed(Amaranthus retroflexus L.)populations from China[J].Pestic Biochem Physiol,2015,124:43-47.
[36]WERLE R,BEGCY K,YERKA M K,et al.Independent evolution of acetolactate synthase-inhibiting herbicide resistance in weedy Sorghum populations across common geographic regions[J].Weed Sci,2017,65(1):164-176.
[37]HILL C M,DUGGLEBY R G.Mutagenesis of Escherichia coli acetohydroxyacid synthase isoenzymeⅡand characterization of three herbicide-insensitive forms[J].Biochem J,1998,335(3):653-661.
[38]DUGGLEBY R G,PANG S S,YU H Q,et al.Systematic characterization of mutations in yeast acetohydroxyacid synthase[J].FEBS J,2003,270(13):2895-2904.
[39]JUNG S M,LE D T,YOON S S,et al.Amino acids conferring herbicide resistance in tobacco acetohydroxyacid synthase[J].Bio J,2014,383:53-61.