定点突变鉴定Myxococcus sp.V11麦芽糖基海藻糖水解酶MTHase的催化中心  

Identification of Amino Acids Related to Catalytic Function of Myxococcus sp.V11 Maltooligosyltrehalose Trehalohydrolase by Site-Directed Mutagenesis

作　　者：陈允妲 李雪亮 吴映函 赵晓艳[1] 吴晓玉[1] 王飞[1] 陈金印 CHEN Yunda;LI Xueliang;WU Yinghan;ZHAO Xiaoyan;WU Xiaoyu;WANG Fei;CHEN Jinyin(Jiangxi Engineering Laboratory for the Development and Utilization of Agricultural Microbial Resources,College of Bioscience and Bioengineering,Jiangxi Agricultural University,Nanchang 330045,China;Collaborative Innovation Center of Postharvest Key Technology and Quality Safety of Fruits and Vegetables in Jiangxi Province,Nanchang 330045,China)

机构地区：[1]江西省农业微生物资源开发与利用工程实验室,江西农业大学生物科学与工程学院,江西南昌330045 [2]江西省果蔬保鲜与质量安全创新中心,江西南昌330045

出　　处：《江西农业大学学报》2021年第1期190-197,共8页Acta Agriculturae Universitatis Jiangxiensis

基　　金：国家自然科学基金项目(31560031);江西省教育厅科学技术研究项目(GJJ160387)。

摘　　要：【目的】麦芽寡糖基海藻糖水解酶(MTHase)是一种水解与α-1,1糖苷键相邻的α-1,4糖苷键,生成麦芽寡糖和海藻糖,是以淀粉为底物生产海藻糖工艺中的关键酶。实验室从菌株Myxococcus sp.V11基因组中克隆到麦芽寡糖基海藻糖水解酶基因并异源表达,通过氨基酸序列分析和三维建模,推测其可能的催化中心氨基酸残基构成。【方法】通过对MTHase上可能存在的3个活性位点D256、E291、D386进行PCR介导定点突变,分别将其突变成为D256A、E291A和D386A。【结果】系统进化树结果表明Myxococcus sp.V11所产MTHase与已报道的Deinococcus radiodurans所产麦芽寡糖基海藻糖水解酶(Maltooligosyltrehalose Trehalohydrolase,DR0464)相似度最高,但仅为49.23%,与已报道的淀粉酶相似度则有较大差异。氨基酸序列比对结果表明MTHase与其它来源的麦芽寡糖基海藻糖水解酶都具有相同的保守序列:GTFTPEG(113-119)、WGYDG(153-157)、ED(291-292)、GLXVXLDXVXNHXGPXGNY(184-202)、DGXRXDA(251-257)、IQNHDQ(382-387)。三联体活性中心(Asp256-Glu291-Asp386)均位于保守区域之内,其中Asp256是亲核试剂,Glu291是酸碱催化的质子供体,Asp386起着维持Glu291稳定构象的作用。以已解析的相似性最高的麦芽寡糖基海藻糖水解酶(PDB ID:2BHU)为模板,在SWISS-MODEL上通过同源建模得到MTHase的三维结构模型。MTHase与2BHU虽然相似性仅为49%,但都具有一个典型GH13家族糖苷水解酶所共有的(α/β)8桶状结构域。三维结构显示MTHase主要由3个结构域构成:催化结构域Domain A(Ala95-Asp316,Gln372-Ser512)形成一个典型的(α/β)_(8)桶状结构,Domain A的中间形成一个裂口,活性中心位于口袋之内;N端结构域(Domain B)主要为β-折叠结构,Domain C也由β-折叠构成,催化三联体Asp256-Glu291-Asp386位于靠近Domain C的位置。MTHase与底物连接的氨基酸残基Gly197,Pro198,Gly200,Asn201,Tyr202,Trp210,Asp316,Asp317,His320,Tyr337,Arg366,Asn390均与2BHU一[Objective]Maltooligosyltrehalose trehalohydrolase(MTHase)is an a-amylase that hydrolyzes the a-1,4 bond adjacent to the a-1,1 bond of maltooligosyltrehalose to release trehalose.In previous studies,the recombinant enzyme MTHase from Myxococcus sp.V11 we cloned and purified,and the expected amino ac⁃ids related to its catalysis were suggested by a sequence homology search[.Method]For further validating these amino acids in this study,they were modified using site-directed mutagenesis and the activity of the mutant en⁃zymes was examined using spectrophotometric analysis and then estimated by homology modeling[.Result]The amino acid sequence of MTHase shared the highest identity(49.23%)with the maltooligosyltrehalose trehalohy⁃drolase from Deinococcus radiodurans.MTHase also shared an identity with maltooligosyltrehalose trehalohydro⁃lase or alpha-amylase from the genome sequences of other bacterial genera in the GenBank database at a level of about 40%.The results of homology modeling and sequence comparing of MTHase and other maltooligosyltre⁃halose trehalohydrolases revealed that MTHase possessed the common maltooligosyltrehalose trehalohydrolase motifs such as GTFTPEG(amino acid residues 113 to 119),WGYDG(amino acid residues 153 to 157),ED(amino acid residues 291 to 292),GLXVXLDXVXNHXGPXGNY(amino acid residues 184 to 202),DGXRX⁃DA(amino acid residues 251 to 257)and IQNHDQ(amino acid residues 382 to 387).A catalytic triad Asp256-Glu291-Asp386 in MTHase was highly conserved in trehalose synthase.Asp256 acted as the catalytic nucleo⁃phile/base,and Glu291 was the catalytic proton donor.Asp386 was not directly involved in the catalysis but had been proposed to stabilize the transition state and to maintain Glu291 in the correct protonation state.Using maltooligosyltrehalose trehalohydrolase catalyzing maltooligosyltrehalose into trehalose as template(PDB ID:2BHU),a MTHase model was built through SWISS-MODEL.The sequence identity of MTHase and maltooligos⁃yltrehalose trehalohydrolase was 49%,but both

关 键 词：Myxococcus sp.V11 麦芽寡糖基海藻糖水解酶 定点突变 催化中心 

分 类 号：Q556[生物学—生物化学] Q943.2[生物学—植物学]