解淀粉芽胞杆菌WS-8中LanM基因的克隆及生物信息学分析
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摘要
克隆解淀粉芽胞杆菌WS-8 (Bacillus amyloliquefaciens WS-8)中的第二类羊毛硫肽合成酶LanM基因,并对LanM编码蛋白的理化性质及结构特征进行分析。设计LanM基因(登录号:APQ49580.1)扩增引物,提取解淀粉芽胞杆菌WS-8基因组DNA,以其作为模板进行PCR扩增。综合多种软件预测和分析LanM编码蛋白的理化性质、结构域和二级结构等。采用邻位连接法(Neighbor-Joining, NJ)构建系统发育树来分析LanM所编码蛋白与同源蛋白的亲缘关系,以及各蛋白结构域的亲缘关系。PCR扩增出的目的条带约为2 840 bp,通过测序鉴定,序列信息与基因组数据一致。LanM基因编码961个氨基酸,等电点为6.07,相对分子质量为111.485 1 kD。LanM编码的蛋白属于亲水性蛋白,无信号肽。该蛋白含有LANC_like结构域,其二级结构主要由螺旋结构和环状结构组成。系统发育树的分析结果表明LANC_like结构域和LanM同源蛋白的亲缘关系一致。解淀粉芽胞杆菌WS-8中存在第二类羊毛硫肽合成酶LanM基因。揭示了羊毛硫肽合成酶LanM发挥脱水和环化作用的理化性质和结构基础,以期为进一步阐明羊毛硫肽合成酶的生物学功能提供参考。
The study was aimed to clone the LanM gene of type Ⅱ lanthipeptide synthetases from Bacillus amyloliquefaciens WS-8 and to analyze the physicochemical property and structure characteristics of LanM encoded protein. Primer for LanM gene(Accession: APQ49580.1) amplification was designed to extract genome DNA from B. amyloliquefaciens WS-8, which was later taken as template for PCR amplification. The physicochemical properties, function domains and secondary structures of LanM encoded protein were analyzed and predicted by various software. Neighbor-Joining method(NJ) was applied to construct the phylogenetic trees so as to analyze the genetic relationship between LanM encoded proteins and homologous proteins, and the genetic relationship of their function domains. The size of the target band amplified by PCR was about 2 840 bp, of which the sequencing results were consistent with the genome data. LanM gene encoded 961 amino acids and the relativemolecular mass was 111.485 1 kD, with the isoelectric point of 6.07. LanM encoded protein belonged to the hydrophilic protein. The encoded protein had a LANC_like domain but no signal peptide, and the helical structure and the cyclic structure dominated secondary structure of the encoded protein. The phylogenetic trees analysis indicated that the genetic relationship of LANC_like domains were consistent to LanM homologous proteins. There is a LanM gene of type Ⅱ lanthipeptide synthetases in B. amyloliquefaciens WS-8. The study revealed the physicochemical properties and structural basis of lanthipeptide synthetases for dehydration and cyclization, which would provide reference for further elucidating the biological function of lanthipeptide synthetases.
The study was aimed to clone the LanM gene of type Ⅱ lanthipeptide synthetases from Bacillus amyloliquefaciens WS-8 and to analyze the physicochemical property and structure characteristics of LanM encoded protein. Primer for LanM gene(Accession: APQ49580.1) amplification was designed to extract genome DNA from B. amyloliquefaciens WS-8, which was later taken as template for PCR amplification. The physicochemical properties, function domains and secondary structures of LanM encoded protein were analyzed and predicted by various software. Neighbor-Joining method(NJ) was applied to construct the phylogenetic trees so as to analyze the genetic relationship between LanM encoded proteins and homologous proteins, and the genetic relationship of their function domains. The size of the target band amplified by PCR was about 2 840 bp, of which the sequencing results were consistent with the genome data. LanM gene encoded 961 amino acids and the relativemolecular mass was 111.485 1 kD, with the isoelectric point of 6.07. LanM encoded protein belonged to the hydrophilic protein. The encoded protein had a LANC_like domain but no signal peptide, and the helical structure and the cyclic structure dominated secondary structure of the encoded protein. The phylogenetic trees analysis indicated that the genetic relationship of LANC_like domains were consistent to LanM homologous proteins. There is a LanM gene of type Ⅱ lanthipeptide synthetases in B. amyloliquefaciens WS-8. The study revealed the physicochemical properties and structural basis of lanthipeptide synthetases for dehydration and cyclization, which would provide reference for further elucidating the biological function of lanthipeptide synthetases.
引文
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Dong S.H.,Tang W.,Lukk T.,Yu Y.,Nair S.K.,and Donk W.A.,2015,The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold,Elife,4:e07607
Garg V.K.,Avashthi H.,Tiwari A.,Jain P.A.,Ramkete P.W.,Kayastha A.M.,and Singh V.K.,2016,Open access MFP-PI-Multi FASTA Prot Param interface,Bioinformation,12(2):74-77
Hu Y.T.,An L.K.,Yin S.L.,Cheng H.C.,Zhang G.W.,Zhang L.P.,and Liu H.W.,2016,Bioinformatics to predict typeⅣlanthipeptide synthetases,Weishengwuxue Tongbao(Microbiology China),43(11):2464-2472(胡彦婷,安丽康,尹淑丽,程辉彩,张根伟,张丽萍,刘洪伟,2016,推定第四类羊毛硫肽合成酶生物信息学分析,微生物学通报,43(11):2464-2472)
Huang C.,Chen M.,Pang D.J.,Bi D.D.,Zou Y.,Xia X.Q.,Yang W.W.,Lou L.P.,Deng R.K.,Tan H.L.,Zhou L.,Yu S.Y.,Guo L.H.,Du X.X.,Cui Y.Y.,Hu J.H.,Mao Q.,Worley P.F.,and Xiao B.,2014,Developmental and activity-dependent expression of LanCL1 confers antioxidant activity required for neuronal survival,Developmental Cell,30(4):479-487
Iftime D.,Jasyk M.,Kulik A.,Imhoff J.F.,Steqmann E.,Wohlleben W.,Süssmuth R.D.,and Weber T.,2015,Streptocollin,a Type IV Lanthipeptide Produced by Streptomyces collinus Tü365,Chembiochem,16(18):2615-2623
Letunic I.,Doerks T.,and Bork P.,2012,SMART 7:recent updates to the protein domain annotation resource,Nucleic Acids Res.,40(D1):D302-D305
Ma H.C.,Gao Y.,Zhao F.Y.,and Zhong J.,2015,Individual catalytic activity of two functional domains of bovicin HJ50synthase BovM,Weishengwu Xuebao(Acta Microbiologica Sinica),55(1):50-58(马宏初,高涌,赵方圆,钟瑾,2015,羊毛硫细菌素bovicin HJ50修饰酶Bov M双功能域单独催化活性鉴定,微生物学报,55(1):50-58)
Mcguffin L.J.,Bryson K.,and Jones D.T.,2000,The PSIPREDprotein structure prediction server,Bioinformatics,16(4):404
Mo T.L.,Xue L.G.,and Zhang Q.,2016,Recent advances in lanthipeptide biosynthesis-A review,Weishengwu Xuebao(Acta Microbiologica Sinica),56(3):373-382(莫天录,薛林贵,张琪,2016,羊毛硫肽类化合物生物合成新进展,微生物学报,56(3):373-382)
Molloy E.M.,Ross R.P.,and Hill C.,2012,'Bac'to the future:bioengineering lantibiotics for designer purposes,Biochemical Society Transactions,40(6):1492-1497
Nishie M.,Nagao J.,and Sonomoto K.,2012,Antibacterial peptides“bacteriocins”:an overview of their diverse characteristics and applications,Biocontrol Science,17(1):1-16
Paul M.,Patton G.C.,and Donk W.A.V.D.,2007,Mutants of the zinc ligands of lacticin 481 synthetase retain dehydration activity but have impaired cyclization activity,Biochemistry,46(21):6268-6276
van der Donk W.A.,and Nair S.K.,2014,Structure and mechanism of lanthipeptide biosynthetic enzymes,Current Opinion in Structural Biology,29:58-66
Zamani M.,Nezafat N.,Negahdaripour M.,Dabbagh F.,and Ghasemi Y.,2015,In silico evaluation of different signal peptides for the secretory production of human growth hormone in E.coli,International Journal of Peptide Research and Therapeutics,21(3):261-268
Zhang Q.,Yu Y.,Vélasquez J.E.,and Van W.A.,2012,Evolution of lanthipeptide synthetases,Proc.NatI.Acad.Sci.USA,109(45):18361
Zhao D.L.,Li J.Y.,Fang S.M.,Wei J.S.,Wang W.,and Liang X.L.,2015,The bioinformatics analysis of Prp5 proteins in some model fungi,Weishengwuxue Tongbao(Microbiology China),42(8):1458-1465(赵东磊,李金玉,方淑梅,韦江司,王伟,梁喜龙,2015,部分模式真菌中Prp5蛋白生物信息学分析,微生物学通报,42(8):1458-1465)
Zhou X.G.,Hou S.M.,Chen D.W.,Tao N.,Ding Y.N.,Sun M.L.,and Zhang S.S.,2011,Genome-wide analysis of the secreted proteins of phytophthora infestans,Hereditas,33(7):785-793
Dong S.H.,Tang W.,Lukk T.,Yu Y.,Nair S.K.,and Donk W.A.,2015,The enterococcal cytolysin synthetase has an unanticipated lipid kinase fold,Elife,4:e07607
Garg V.K.,Avashthi H.,Tiwari A.,Jain P.A.,Ramkete P.W.,Kayastha A.M.,and Singh V.K.,2016,Open access MFP-PI-Multi FASTA Prot Param interface,Bioinformation,12(2):74-77
Hu Y.T.,An L.K.,Yin S.L.,Cheng H.C.,Zhang G.W.,Zhang L.P.,and Liu H.W.,2016,Bioinformatics to predict typeⅣlanthipeptide synthetases,Weishengwuxue Tongbao(Microbiology China),43(11):2464-2472(胡彦婷,安丽康,尹淑丽,程辉彩,张根伟,张丽萍,刘洪伟,2016,推定第四类羊毛硫肽合成酶生物信息学分析,微生物学通报,43(11):2464-2472)
Huang C.,Chen M.,Pang D.J.,Bi D.D.,Zou Y.,Xia X.Q.,Yang W.W.,Lou L.P.,Deng R.K.,Tan H.L.,Zhou L.,Yu S.Y.,Guo L.H.,Du X.X.,Cui Y.Y.,Hu J.H.,Mao Q.,Worley P.F.,and Xiao B.,2014,Developmental and activity-dependent expression of LanCL1 confers antioxidant activity required for neuronal survival,Developmental Cell,30(4):479-487
Iftime D.,Jasyk M.,Kulik A.,Imhoff J.F.,Steqmann E.,Wohlleben W.,Süssmuth R.D.,and Weber T.,2015,Streptocollin,a Type IV Lanthipeptide Produced by Streptomyces collinus Tü365,Chembiochem,16(18):2615-2623
Letunic I.,Doerks T.,and Bork P.,2012,SMART 7:recent updates to the protein domain annotation resource,Nucleic Acids Res.,40(D1):D302-D305
Ma H.C.,Gao Y.,Zhao F.Y.,and Zhong J.,2015,Individual catalytic activity of two functional domains of bovicin HJ50synthase BovM,Weishengwu Xuebao(Acta Microbiologica Sinica),55(1):50-58(马宏初,高涌,赵方圆,钟瑾,2015,羊毛硫细菌素bovicin HJ50修饰酶Bov M双功能域单独催化活性鉴定,微生物学报,55(1):50-58)
Mcguffin L.J.,Bryson K.,and Jones D.T.,2000,The PSIPREDprotein structure prediction server,Bioinformatics,16(4):404
Mo T.L.,Xue L.G.,and Zhang Q.,2016,Recent advances in lanthipeptide biosynthesis-A review,Weishengwu Xuebao(Acta Microbiologica Sinica),56(3):373-382(莫天录,薛林贵,张琪,2016,羊毛硫肽类化合物生物合成新进展,微生物学报,56(3):373-382)
Molloy E.M.,Ross R.P.,and Hill C.,2012,'Bac'to the future:bioengineering lantibiotics for designer purposes,Biochemical Society Transactions,40(6):1492-1497
Nishie M.,Nagao J.,and Sonomoto K.,2012,Antibacterial peptides“bacteriocins”:an overview of their diverse characteristics and applications,Biocontrol Science,17(1):1-16
Paul M.,Patton G.C.,and Donk W.A.V.D.,2007,Mutants of the zinc ligands of lacticin 481 synthetase retain dehydration activity but have impaired cyclization activity,Biochemistry,46(21):6268-6276
van der Donk W.A.,and Nair S.K.,2014,Structure and mechanism of lanthipeptide biosynthetic enzymes,Current Opinion in Structural Biology,29:58-66
Zamani M.,Nezafat N.,Negahdaripour M.,Dabbagh F.,and Ghasemi Y.,2015,In silico evaluation of different signal peptides for the secretory production of human growth hormone in E.coli,International Journal of Peptide Research and Therapeutics,21(3):261-268
Zhang Q.,Yu Y.,Vélasquez J.E.,and Van W.A.,2012,Evolution of lanthipeptide synthetases,Proc.NatI.Acad.Sci.USA,109(45):18361
Zhao D.L.,Li J.Y.,Fang S.M.,Wei J.S.,Wang W.,and Liang X.L.,2015,The bioinformatics analysis of Prp5 proteins in some model fungi,Weishengwuxue Tongbao(Microbiology China),42(8):1458-1465(赵东磊,李金玉,方淑梅,韦江司,王伟,梁喜龙,2015,部分模式真菌中Prp5蛋白生物信息学分析,微生物学通报,42(8):1458-1465)
Zhou X.G.,Hou S.M.,Chen D.W.,Tao N.,Ding Y.N.,Sun M.L.,and Zhang S.S.,2011,Genome-wide analysis of the secreted proteins of phytophthora infestans,Hereditas,33(7):785-793