纳豆芽孢杆菌MenA的生物信息学分析及亚细胞定位  

Bioinformatics Analysis and Subcellular Localization of MenA in Bacillus subtilis natto

作　　者：丁秀敏 薛正莲 王洲 杨自名 冯静静 吴佳雯 胡润 章宁娟 刘艳 Ding Xiumin;Xue Zhenglian;Wang Zhou;Yang Ziming;Feng Jingjing;Wu Jiawen;Hu Run;Zhang Ningjuan;Liu Yan(College of Biological and Chemical Engineering,Anhui Ploytechnic University,Wuhu,241000)

机构地区：[1]安徽工程大学生物与化学工程学院,芜湖241000

出　　处：《基因组学与应用生物学》2020年第10期4604-4613,共10页Genomics and Applied Biology

基　　金：国家自然科学基金(31871781,31501465);安徽高校自然科学研究重点项目(KJ2018A0106);安徽省自然科学基金青年基金(1608085QC54);国家级大学生创新创业项目(2016103630055);安徽省大学生创新创业计划项目(20171036-3178);安徽工程大学青年基金(2017YQ01)共同资助。

摘　　要：为了深入研究纳豆芽孢杆菌中1,4-二羟基-2-萘甲酸聚异戊二烯转移酶(BsnMenA)的结构和功能、催化作用机制以及亚细胞定位,本研究首先利用生物信息学软件对BsnMenA的系统进化关系、基本性质、亚细胞定位、结构域和motifs以及空间结构等进行预测。另外还构建了纳豆芽孢杆菌menA和egfp荧光标签的重组菌MEP28和EMP28,通过IPTG诱导表达后,利用激光共聚焦显微镜观察重组菌中荧光所在的位置。系统进化树显示,芽孢杆菌属中的MenA高度相似,亲缘关系较近,但与大肠杆菌等其它科中的MenA亲缘关系较远。从理化性质及亚细胞定位预测,初步确定BsnMenA属于膜结合型蛋白质。结构域和motifs预测显示,BsnMenA的34-278aa组成典型的UbiA结构功能域,其中74-84aa和204-212aa分别存在NxxxDxxxxxD和NNxxDxxxD的Asp-rich motifs结构。通过折叠识别建模法,模拟出BsnMenA的三维空间结构,是由9个跨膜螺旋组成的一个活性中心腔,其中Asp-rich motifs位于胞质区loop的H23和H67中,这与匹配度最高的嗜热系古生细菌中的4-羟苯甲酸-聚异戊二烯转移酶(ApUbiA)极其相似,由于BsnMenA与ApUbiA空间结构高度相似,因此推导出BsnMenA的催化功能有关的保守结构域位点及其催化机制。对重组菌诱导表达20 min后取样,通过激光共聚焦显微镜检测发现,在细菌的细胞膜上有明显可见荧光。通过生物信息学预测以及亚细胞定位实验不仅验证BsnMenA是膜蛋白酶,而且说明BsnMenA能在大肠杆菌中异源表达,这些理论为BsnMenA的深入研究提供理论基础,也为其它未知晶体结构酶的研究提供了有效的研究思路和方法。In order to study the structure,function,catalysis mechanism and subcellular localization of 1,4-dihydroxy-2-naphthoate octaprenyltransferase(BsnMenA)in Bacillus subtilis natto.Bioinformation softwares were used to predict the phylogenetic relationship,basic property,subcellular localization,structural domain and motifs searching,space structure of BsnMenA.In addition,the recombinant bacteria,MEP28 and EMP28,with overexpressedmenA and egfp fluorescence tags were also constructed.After induced by IPTG,the fluorescence location in the recombinant bacteria was observed by confocal laser microscopy.From the perspective of phylogenetic tree,MenAs in Bacillus subtilis were highly similar to each other,but far from other families,such as Escherichia coli.According to prediction of the physical,chemical properties and subcellular localization of the enzyme,it was found that BsnMenA was a kind of intermembrane protein.Structural domain and motif searching showed that there was a highly conserved UbiA structural domain in BsnMenA amino acid sequences from 34 to 278.Moreover,there were two Asp-rich motifs,NxxxDxxxxxD and NNxxDxxxD,from 74 to 84 and from 204 to 212.Through the method of folding identification modeling,three-dimensional space structure of BsnMenA was constructed,and there were 9 transmembrane helixes forming an active central cavity which was embedded in the phospholipid bilayer.The Asp-rich motifs were located at HL23 and HL67 in the cytoplasmic loop,which were very similar with 4-hydroxybenzoate Octaprenyltransferase(ApUbiA)in Aeropyrum pernix.Since the space structures of BsnMenA and ApUbiA are highly similar,the conserved domain sites related to the catalytic function of BsnMenA and its catalytic mechanism are deduced.Smples were taken about 20 min after inducing expression of recombinant bacteria.Confocal laser microscopy showed obviously fluorescence in the cell membrane of the bacteria.According to bioinformatics prediction and subcellular localization experiments,BsnMenA was not only identified as mem

关 键 词：纳豆芽孢杆菌(Bacillus SUBTILIS natto) 异戊烯转移酶MenA 生物信息学分析 亚细胞定位 

分 类 号：Q811.4[生物学—生物工程] Q936