High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases
文献类型：期刊论文
| 作者 | Cui, Gu-Zhen1,2 ; Wang, Shan1,2; Li, Yifei1; Tian, Yi-Jun1; Feng, Yingang1; Cui, Qiu1
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| 刊名 | PROTEIN JOURNAL
 |
| 出版日期 | 2012-06-01 |
| 卷号 | 31期号:5页码:432-438 |
| 关键词 | Cis-epoxysuccinic acid hydrolase Epoxide hydrolase Enzyme kinetics Protein characterization Recombinant expression Homology modeling |
| 中文摘要 | The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form D(-)-tartaric acid or L(?)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[D] and CESH[L], producing D(-)- and L(?)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver– Burk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[L] showed much higher catalytic efficiency than CESH[D], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[L] and CESH[D] have different structural folds and potential active site residues. CESH[L] adopted a typical a/b-hydrolase fold with a cap domain and a core domain, whereas CESH[D] possessed a unique TIM barrel fold composed of 8 a-helices and 8 bstrands, and 2 extra short a-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[D], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs. |
| 英文摘要 | The cis-epoxysuccinate hydrolases (CESHs), members of epoxide hydrolase, catalyze cis-epoxysuccinic acid hydrolysis to form d(-)-tartaric acid or l(+)-tartaric acid which are important chemicals with broad scientific and industrial applications. Two types of CESHs (CESH[d] and CESH[l], producing d(-)- and l(+)-tartaric acids, respectively) have been reported with low yield and complicated purification procedure in previous studies. In this paper, the two CESHs were overexpressed in Escherichia coli using codon-optimized genes. High protein yields by one-step purifications were obtained for both recombinant enzymes. The optimal pH and temperature were measured for both recombinant CESHs, and the properties of recombinant enzymes were similar to native enzymes. Kinetics parameters measured by Lineweaver-Burk plot indicates both enzymes exhibited similar affinity to cis-epoxysuccinic acid, but CESH[l] showed much higher catalytic efficiency than CESH[d], suggesting that the two CESHs have different catalytic mechanisms. The structures of both CESHs constructed by homology modeling indicated that CESH[l] and CESH[d] have different structural folds and potential active site residues. CESH[l] adopted a typical alpha/beta-hydrolase fold with a cap domain and a core domain, whereas CESH[d] possessed a unique TIM barrel fold composed of 8 alpha-helices and 8 beta-strands, and 2 extra short alpha-helices exist on the top and bottom of the barrel, respectively. A divalent metal ion, preferred to be zinc, was found in CESH[d], and the ion was proved to be crucial to the enzymatic activity. These results provide structural insight into the different catalytic mechanisms of the two CESHs. |
| WOS标题词 | Science & Technology ; Life Sciences & Biomedicine |
| 学科主题 | 代谢物组学 |
| 类目[WOS] | Biochemistry & Molecular Biology |
| 研究领域[WOS] | Biochemistry & Molecular Biology |
| 关键词[WOS] | BURKHOLDERIA-CEPACIA MBA4 ; EPOXIDE HYDROLASES ; MULTIPLE TEMPLATES ; CRYSTAL-STRUCTURES ; ENZYME ; DEHALOGENASE ; CLONING ; FOLD ; GENE |
| 收录类别 | SCI |
| 语种 | 英语 |
| WOS记录号 | WOS:000305531100010 |
| 公开日期 | 2014-03-26 |
| 源URL | [http://ir.qibebt.ac.cn:8080/handle/337004/1722]  |
| 专题 | 青岛生物能源与过程研究所_代谢物组学团队 |
| 作者单位 | 1.Chinese Acad Sci, Shandong Prov Key Lab Energy Genet, Key Lab Biofuels, Qingdao Inst Bioenergy & Bioproc Technol, Qingdao 266101, Peoples R China 2.Chinese Acad Sci, Grad Sch, Beijing 100049, Peoples R China |
| 推荐引用方式 GB/T 7714 | Cui, Gu-Zhen,Wang, Shan,Li, Yifei,et al. High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases[J]. PROTEIN JOURNAL,2012,31(5):432-438. |
| APA | Cui, Gu-Zhen,Wang, Shan,Li, Yifei,Tian, Yi-Jun,Feng, Yingang,&Cui, Qiu.(2012).High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases.PROTEIN JOURNAL,31(5),432-438. |
| MLA | Cui, Gu-Zhen,et al."High Yield Recombinant Expression, Characterization and Homology Modeling of Two Types of Cis-epoxysuccinic Acid Hydrolases".PROTEIN JOURNAL 31.5(2012):432-438. |
入库方式： OAI收割
来源：青岛生物能源与过程研究所
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