定点突变提高N-酰基高丝氨酸内酯酶酶活和温度稳定性
|
摘要
【目的】提高N-酰基高丝氨酸内酯酶(N-acylhomoserine lactonase,AiiA)酶活及温度稳定性。【方法】本研究基于AiiA同源蛋白的三维结构对AiiA进行定点突变,分析野生型AiiA及其突变蛋白酶活和温度稳定性。【结果】野生型AiiA较不稳定,在45℃下温浴30 min,或4℃储存5 d后均失去降解N-酰基高丝氨酸内酯(N-acylhomoserine lactone,AHL)的活性。但是突变AiiA蛋白(N65K,T195R和A206E)的酶活力较野生型AiiA均提高了20%以上,且4℃储存时间延长到7 d。此外,突变株N65K比野生型AiiA对高温具有更强的耐受性,在45℃温浴后剩余酶活力达到45%以上,55℃温浴30 min后仍保留5.0%的酶活力。【结论】通过定点突变改造AiiA蛋白结构,提升了AiiA蛋白的酶活和温度稳定性。
[Objective] To enhance enzymatic activity and thermostability of N-acylhomoserine lactonase( AiiA).[Methods] We performed site-directed mutagenesis based on AiiA homologous 3-D protein structure,and analyzed enzymatic activity and thermostability of both wild type and mutated AiiA. [Results] The wild type AiiA lost its Nacylhomoserine lactone( AHL) degrading activity after being incubated at 45℃ for 30 min or after being stored at 4℃ for5 days. By comparison,the AHL-degrading activities of three types of mutated AiiA( N65K,T195R,and A206E) were enhanced,and their storage periods at 4℃ were extended to 7 days. In addition,the N65K mutant acquired higher temperature tolerance with remain of more than 45% of its enzymatic activity after being incubated at 45℃ and 5. 0%enzymatic activity after being incubated at 55℃ as compared to the wild type. [Conclusion] Molecular modulation by site-directed mutagenesis could significantly improve enzymatic activity and thermostability of AiiA.
[Objective] To enhance enzymatic activity and thermostability of N-acylhomoserine lactonase( AiiA).[Methods] We performed site-directed mutagenesis based on AiiA homologous 3-D protein structure,and analyzed enzymatic activity and thermostability of both wild type and mutated AiiA. [Results] The wild type AiiA lost its Nacylhomoserine lactone( AHL) degrading activity after being incubated at 45℃ for 30 min or after being stored at 4℃ for5 days. By comparison,the AHL-degrading activities of three types of mutated AiiA( N65K,T195R,and A206E) were enhanced,and their storage periods at 4℃ were extended to 7 days. In addition,the N65K mutant acquired higher temperature tolerance with remain of more than 45% of its enzymatic activity after being incubated at 45℃ and 5. 0%enzymatic activity after being incubated at 55℃ as compared to the wild type. [Conclusion] Molecular modulation by site-directed mutagenesis could significantly improve enzymatic activity and thermostability of AiiA.
引文
[1]Yeon KM,Cheong WS,Oh HS,Lee WN,Hwang BK,Lee CH,Beyenal H,Lewandowski Z.Quorum sensing:a new biofouling control paradigm in a membrane bioreactor for advanced wastewater treatment.Environmental Science&Technology,2008,43(2):380-385.
[2]Abbas A,Adams C,Scully N,Glennon J,O'Gara F.A role for TonB1 in biofilm formation and quorum sensing in Pseudomonas aeruginosa.FEMS Microbiology Letters,2007,274(2):269-278.
[3]Vikstrm E,Tafazoli F,Magnusson KE.Pseudomonas aeruginosa quorum sensing molecule N-(3oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells.FEBS Letters,2006,580(30):6921-6928.
[4]Antunes LCM,Schaefer AL,Ferreira RB,Qin N,Stevens AM,Ruby EG,Greenberg EP.Transcriptome analysis of the Vibrio fischeri LuxR-LuxI regulon.Journal of Bacteriology,2007,189(22):8387-8391.
[5]Stevens AM,Greenberg E.Quorum sensing in Vibrio fischeri:essential elements for activation of the luminescence genes.Journal of Bacteriology,1997,179(2):557-562.
[6]Nackerdien ZE,Keynan A,Bassler BL,Lederberg J,Thaler DS.Quorum sensing influences Vibrio harveyi growth rates in a manner not fully accounted for by the marker effect of bioluminescence.PloS One,2008,3(2):e1671.
[7]Pompeani AJ,Irgon JJ,Berger MF,Bulyk ML,Wingreen NS,Bassler BL.The Vibrio harveyi master quorumsensing regulator,LuxR,a TetR-type protein is both an activator and a repressor:DNA recognition and binding specificity at target promoters.Molecular Microbiology,2008,70(1):76-88.
[8]Cho H,Winans SC.TraA,TraC and TraD autorepress two divergent quorum-regulated promoters near the transfer origin of the Ti plasmid of Agrobacterium tumefaciens.Molecular Microbiology,2007,63(6):1769-1782.
[9]Lowe N,Gan HM,Chakravartty V,Scott R,Szegedi E,Burr TJ,Savka MA.Quorum-sensing signal production by Agrobacterium vitis strains and their tumor-inducing and tartrate-catabolic plasmids.FEMS Microbiology Letters,2009,296(1):102-109.
[10]Barnard AM,Bowden SD,Burr T,Coulthurst SJ,Monson RE,Salmond GP.Quorum sensing,virulence and secondary metabolite production in plant soft-rotting bacteria.Philosophical Transactions of the Royal Society B:Biological Sciences,2007,362(1483):1165-1183.
[11]Zhao C,Zeng H,Yu Z,Sun M.N-Acyl homoserine lactonase promotes prevention of Erwinia virulence with Zwittermicin A-producing strain Bacillus cereus.Biotechnology and Bioengineering,2008,100(3):599-603.
[12]De Kievit TR,Iglewski BH.Bacterial quorum sensing in pathogenic relationships.Infection and Immunity,2000,68(9):4839-4849.
[13]Dong YH,Gusti AR,Zhang Q,Xu JL,Zhang LH.Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species.Applied and Environmental Microbiology,2002,68(4):1754-1759.
[14]Lee SJ,Park SY,Lee JJ,Yum DY,Koo BT,Lee JK.Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis.Applied and Environmental Microbiology,2002,68(8):3919-3924.
[15]Van Kessel JC,Ulrich LE,Zhulin IB,Bassler BL.Analysis of activator and repressor functions reveals the requirements for transcriptional control by LuxR,the master regulator of quorum sensing in Vibrio harveyi.mBio,2013,4(4):e00378-13.
[16]Da Silva Araújo FD,Esper LMR,Kuaye AY,Sircili MP,Marsaioli AJ.N-Acyl-homoserine Lactones from Enterobacter sakazakii(Cronobacter spp.)and Their Degradation by Bacillus cereus Enzymes.Journal of Agricultural and Food Chemistry,2012,60(2):585-592.
[17]Mahmoudi E,Naderi D,Venturi V.AiiA lactonase disrupts N-acylhomoserine lactone and attenuates quorumsensing-related virulence in Pectobacterium carotovorum EMPCC.Annals of Microbiology,2013,63:691-697.
[18]Park SY,Lee SJ,Oh TK,Oh JW,Koo BT,Yum DY,Lee JK.AhlD,an N-acylhomoserine lactonase in Arthrobacter sp.,and predicted homologues in other bacteria.Microbiology,2003,149(6):1541-1550.
[19]Wang WZ,Morohoshi T,Ikenoya M,Someya N,Ikeda T.AiiM,a novel class of N-acylhomoserine lactonase from the leaf-associated bacterium Microbacterium testaceum.Applied and Environmental Microbiology,2010,76(8):2524-2530.
[20]Wang WZ,Morohoshi T,Someya N,Ikeda T.AidC,a novel N-acylhomoserine lactonase from the potato rootassociated Cytophaga-Flavobacteria-Bacteroides(CFB)group bacterium Chryseobacterium sp.strain StRB126.Applied and Environmental Microbiology,2012,78(22):7985-7992.
[21]Yang M,Zhang F,Lin B,Su X,Guo L,Huang Z,Guan X.Soluble expression of AiiA protein and analysis of its antimicrobial activity.Journal of Molecular Cell Biology,2008,41(6):465-472.(in Chinese)杨梅,张锋,林彬辉,苏新华,郭丽清,黄志鹏,关雄.AiiA蛋白的可溶性表达及其抗菌活性研究.分子细胞生物学报,2008,41(6):465-472.
[22]Liu D,Momb J,Thomas PW,Moulin A,Petsko GA,Fast W,Ringe D.Mechanism of the Quorum-Quenching Lactonase(AiiA)from Bacillus thuringiensis.1.ProductBound Structures.Biochemistry,2008,47(29):7706-7714.
[23]Arnold K,Bordoli L,Kopp J,Schwede T.The SWISSMODEL workspace:a web-based environment for protein structure homology modelling.Bioinformatics,2006,22(2):195-201.
[24]Schwede T,Kopp J,Guex N,Peitsch MC.SWISSMODEL:an automated protein homology-modeling server.Nucleic Acids Research,2003,31(13):3381-3385.
[25]Sambrook J,Russell DW.Molecular Cloning:A Laboratory Manua.3rdeds.NewYork:Cold Spring Laboratory Press,2001.
[26]Zor T,Selinger Z.Linearization of the Bradford protein assay increases its sensitivity:theoretical and experimental studies.Analytical Biochemistry,1996,236(2):302-308.
[27]Mabrouk SB,Aghajari N,Ali MB,Messaoud EB,Juy M,Haser R,Bejar S.Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions.Bioresource Technology,2011,102(2):1740-1746.
[28]Zhang SB,Wu ZL.Identification of amino acid residues responsible for increased thermostability of feruloyl esterase A from Aspergillus niger using the PoPMuSiC algorithm.Bioresource Technology,2011,102(2):2093-2096.
[29]Wang Z,Chen T,Ma X,Shen Z,Zhao X.Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum.Bioresource Technology,2011,102(4):3934-3940.
[30]Bunting K,Cooper J,Badasso MO,Tickle IJ,Newton M,Wood SP,Zhang Y,Young D.Engineering a change in metal-ion specificity of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis.European Journal of Biochemistry,1998,251(3):795-803.
[31]Chen Y,Chou W,Chang G.Characterization of the interactions between Asp141 and Phe236 in the Mn2+-Lmalate binding of pigeon liver malic enzyme.Journal of Biochemistry,2003,374:633-637.
[32]Obradors N,Cabiscol E,Aguilar J,Ros J.Site-directed mutagenesis studies of the metal-binding center of the iron-dependent propanediol oxidoreductase from Escherichia coli.European Journal of Biochemistry,1998,258(1):207-213.
[33]Yin J,Shan L,Song D,Zhong J.Site-directed mutagenesis of the zinc-binding domain of Trichoplusia ni granulovirus enhancin.Acta Entomologica Sinica,2007,50(11):1111-1115.(in Chinese)尹隽,单梁,宋大新,钟江.粉纹夜蛾颗粒体病毒增强蛋白锌离子结合域定点突变.昆虫学报,2007,50(11):1111-1115.
[34]Qiu J,Li C,Jia Z,Zhang X,Ma H,Song S.Enzymatic characterization and function of N-acylhomoserine lactonase SS10.Acta Phytopathol Sinica,2007,37(6):629-636.(in Chinese)邱健,李承光,贾振华,张霞,马宏,宋水山.酰基高丝氨酸内酯酶SS10的酶学特性及其抗软腐病功能的初探.植物病理学报,2007,37(6):629-636.
[35]Watanabe K,Chishiro K,Kitamura K,Suzuki Y.Proline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006.Journal of Biological Chemistry,1991,266(36):24287-24294.
[2]Abbas A,Adams C,Scully N,Glennon J,O'Gara F.A role for TonB1 in biofilm formation and quorum sensing in Pseudomonas aeruginosa.FEMS Microbiology Letters,2007,274(2):269-278.
[3]Vikstrm E,Tafazoli F,Magnusson KE.Pseudomonas aeruginosa quorum sensing molecule N-(3oxododecanoyl)-l-homoserine lactone disrupts epithelial barrier integrity of Caco-2 cells.FEBS Letters,2006,580(30):6921-6928.
[4]Antunes LCM,Schaefer AL,Ferreira RB,Qin N,Stevens AM,Ruby EG,Greenberg EP.Transcriptome analysis of the Vibrio fischeri LuxR-LuxI regulon.Journal of Bacteriology,2007,189(22):8387-8391.
[5]Stevens AM,Greenberg E.Quorum sensing in Vibrio fischeri:essential elements for activation of the luminescence genes.Journal of Bacteriology,1997,179(2):557-562.
[6]Nackerdien ZE,Keynan A,Bassler BL,Lederberg J,Thaler DS.Quorum sensing influences Vibrio harveyi growth rates in a manner not fully accounted for by the marker effect of bioluminescence.PloS One,2008,3(2):e1671.
[7]Pompeani AJ,Irgon JJ,Berger MF,Bulyk ML,Wingreen NS,Bassler BL.The Vibrio harveyi master quorumsensing regulator,LuxR,a TetR-type protein is both an activator and a repressor:DNA recognition and binding specificity at target promoters.Molecular Microbiology,2008,70(1):76-88.
[8]Cho H,Winans SC.TraA,TraC and TraD autorepress two divergent quorum-regulated promoters near the transfer origin of the Ti plasmid of Agrobacterium tumefaciens.Molecular Microbiology,2007,63(6):1769-1782.
[9]Lowe N,Gan HM,Chakravartty V,Scott R,Szegedi E,Burr TJ,Savka MA.Quorum-sensing signal production by Agrobacterium vitis strains and their tumor-inducing and tartrate-catabolic plasmids.FEMS Microbiology Letters,2009,296(1):102-109.
[10]Barnard AM,Bowden SD,Burr T,Coulthurst SJ,Monson RE,Salmond GP.Quorum sensing,virulence and secondary metabolite production in plant soft-rotting bacteria.Philosophical Transactions of the Royal Society B:Biological Sciences,2007,362(1483):1165-1183.
[11]Zhao C,Zeng H,Yu Z,Sun M.N-Acyl homoserine lactonase promotes prevention of Erwinia virulence with Zwittermicin A-producing strain Bacillus cereus.Biotechnology and Bioengineering,2008,100(3):599-603.
[12]De Kievit TR,Iglewski BH.Bacterial quorum sensing in pathogenic relationships.Infection and Immunity,2000,68(9):4839-4849.
[13]Dong YH,Gusti AR,Zhang Q,Xu JL,Zhang LH.Identification of quorum-quenching N-acyl homoserine lactonases from Bacillus species.Applied and Environmental Microbiology,2002,68(4):1754-1759.
[14]Lee SJ,Park SY,Lee JJ,Yum DY,Koo BT,Lee JK.Genes encoding the N-acyl homoserine lactone-degrading enzyme are widespread in many subspecies of Bacillus thuringiensis.Applied and Environmental Microbiology,2002,68(8):3919-3924.
[15]Van Kessel JC,Ulrich LE,Zhulin IB,Bassler BL.Analysis of activator and repressor functions reveals the requirements for transcriptional control by LuxR,the master regulator of quorum sensing in Vibrio harveyi.mBio,2013,4(4):e00378-13.
[16]Da Silva Araújo FD,Esper LMR,Kuaye AY,Sircili MP,Marsaioli AJ.N-Acyl-homoserine Lactones from Enterobacter sakazakii(Cronobacter spp.)and Their Degradation by Bacillus cereus Enzymes.Journal of Agricultural and Food Chemistry,2012,60(2):585-592.
[17]Mahmoudi E,Naderi D,Venturi V.AiiA lactonase disrupts N-acylhomoserine lactone and attenuates quorumsensing-related virulence in Pectobacterium carotovorum EMPCC.Annals of Microbiology,2013,63:691-697.
[18]Park SY,Lee SJ,Oh TK,Oh JW,Koo BT,Yum DY,Lee JK.AhlD,an N-acylhomoserine lactonase in Arthrobacter sp.,and predicted homologues in other bacteria.Microbiology,2003,149(6):1541-1550.
[19]Wang WZ,Morohoshi T,Ikenoya M,Someya N,Ikeda T.AiiM,a novel class of N-acylhomoserine lactonase from the leaf-associated bacterium Microbacterium testaceum.Applied and Environmental Microbiology,2010,76(8):2524-2530.
[20]Wang WZ,Morohoshi T,Someya N,Ikeda T.AidC,a novel N-acylhomoserine lactonase from the potato rootassociated Cytophaga-Flavobacteria-Bacteroides(CFB)group bacterium Chryseobacterium sp.strain StRB126.Applied and Environmental Microbiology,2012,78(22):7985-7992.
[21]Yang M,Zhang F,Lin B,Su X,Guo L,Huang Z,Guan X.Soluble expression of AiiA protein and analysis of its antimicrobial activity.Journal of Molecular Cell Biology,2008,41(6):465-472.(in Chinese)杨梅,张锋,林彬辉,苏新华,郭丽清,黄志鹏,关雄.AiiA蛋白的可溶性表达及其抗菌活性研究.分子细胞生物学报,2008,41(6):465-472.
[22]Liu D,Momb J,Thomas PW,Moulin A,Petsko GA,Fast W,Ringe D.Mechanism of the Quorum-Quenching Lactonase(AiiA)from Bacillus thuringiensis.1.ProductBound Structures.Biochemistry,2008,47(29):7706-7714.
[23]Arnold K,Bordoli L,Kopp J,Schwede T.The SWISSMODEL workspace:a web-based environment for protein structure homology modelling.Bioinformatics,2006,22(2):195-201.
[24]Schwede T,Kopp J,Guex N,Peitsch MC.SWISSMODEL:an automated protein homology-modeling server.Nucleic Acids Research,2003,31(13):3381-3385.
[25]Sambrook J,Russell DW.Molecular Cloning:A Laboratory Manua.3rdeds.NewYork:Cold Spring Laboratory Press,2001.
[26]Zor T,Selinger Z.Linearization of the Bradford protein assay increases its sensitivity:theoretical and experimental studies.Analytical Biochemistry,1996,236(2):302-308.
[27]Mabrouk SB,Aghajari N,Ali MB,Messaoud EB,Juy M,Haser R,Bejar S.Enhancement of the thermostability of the maltogenic amylase MAUS149 by Gly312Ala and Lys436Arg substitutions.Bioresource Technology,2011,102(2):1740-1746.
[28]Zhang SB,Wu ZL.Identification of amino acid residues responsible for increased thermostability of feruloyl esterase A from Aspergillus niger using the PoPMuSiC algorithm.Bioresource Technology,2011,102(2):2093-2096.
[29]Wang Z,Chen T,Ma X,Shen Z,Zhao X.Enhancement of riboflavin production with Bacillus subtilis by expression and site-directed mutagenesis of zwf and gnd gene from Corynebacterium glutamicum.Bioresource Technology,2011,102(4):3934-3940.
[30]Bunting K,Cooper J,Badasso MO,Tickle IJ,Newton M,Wood SP,Zhang Y,Young D.Engineering a change in metal-ion specificity of the iron-dependent superoxide dismutase from Mycobacterium tuberculosis.European Journal of Biochemistry,1998,251(3):795-803.
[31]Chen Y,Chou W,Chang G.Characterization of the interactions between Asp141 and Phe236 in the Mn2+-Lmalate binding of pigeon liver malic enzyme.Journal of Biochemistry,2003,374:633-637.
[32]Obradors N,Cabiscol E,Aguilar J,Ros J.Site-directed mutagenesis studies of the metal-binding center of the iron-dependent propanediol oxidoreductase from Escherichia coli.European Journal of Biochemistry,1998,258(1):207-213.
[33]Yin J,Shan L,Song D,Zhong J.Site-directed mutagenesis of the zinc-binding domain of Trichoplusia ni granulovirus enhancin.Acta Entomologica Sinica,2007,50(11):1111-1115.(in Chinese)尹隽,单梁,宋大新,钟江.粉纹夜蛾颗粒体病毒增强蛋白锌离子结合域定点突变.昆虫学报,2007,50(11):1111-1115.
[34]Qiu J,Li C,Jia Z,Zhang X,Ma H,Song S.Enzymatic characterization and function of N-acylhomoserine lactonase SS10.Acta Phytopathol Sinica,2007,37(6):629-636.(in Chinese)邱健,李承光,贾振华,张霞,马宏,宋水山.酰基高丝氨酸内酯酶SS10的酶学特性及其抗软腐病功能的初探.植物病理学报,2007,37(6):629-636.
[35]Watanabe K,Chishiro K,Kitamura K,Suzuki Y.Proline residues responsible for thermostability occur with high frequency in the loop regions of an extremely thermostable oligo-1,6-glucosidase from Bacillus thermoglucosidasius KP1006.Journal of Biological Chemistry,1991,266(36):24287-24294.