Cloning and characterization of GDP-perosamine synthetase (Per) from Escherichia coli O157:H7 and synthesis of GDP-perosamine in vitro
Cloning and characterization of GDP-perosamine synthetase (Per) from Escherichia coli O157:H7 and synthesis of GDP-perosamine in vitro
复制标题
大肠杆菌 O157:H7 的 GDP-过胺合成酶 (Per) 的克隆和表征以及 GDP-过胺的体外合成。
DOI：
10.1016/j.bbrc.2007.08.184
复制
发表时间：
2007-11-23
影响因子：
3.1
通讯作者：
Wang, Peng George
中科院分区：
文献类型：
--
作者：
Zhao, Guohul;Liu, Jun;Wang, Peng George
GDP-perosamine合成酶（Per，E.C.尚未分类）对大肠杆菌O 157：H7 O-抗原的合成是重要的。Per基因的突变可破坏O 157 O抗原的合成。本研究从大肠杆菌E. coliO 157：H7中获得高效表达。coli BL21（DE3）。重组His标记的Per融合蛋白为十聚体，分子量为431 kDa。该重组蛋白的最适pH值为7.5。二价离子对Per催化反应没有明显影响。GDP-4-酮-6-脱氧-D-甘露糖的K-m和K-cat/K-m分别为0.09 mM和2.1 x 10（5）M-1 S-1，L-谷氨酸的K-m和K-cat/K-m分别为2 mM和0.52 x 105 M-1 S-1。PER用于从GDP-甘露糖与重组GDP-甘露糖脱氢酶（GMD，E. C. 4.2.1.47）。纯化的GDP-perosamine通过MS和NMR鉴定。本研究为GDP-perosamine的合成提供了一条可行的途径，为致病性大肠杆菌LPS生物合成的研究奠定了基础。coli O157：H7。(C)2007年爱思唯尔公司All rights reserved.
GDP-perosamine synthetase (Per, E.C. not yet classified) is important to the synthesis of Escherichia coli O157 : H7 O-antigen. The mutant in per gene can disrupt the synthesis of O157 O-antigen. In this study, GDP-perosamine synthetase was cloned from E. coli O157 : H7 and over-expressed in E. coli BL21 (DE3). The recombinant His-tagged Per fusion protein was a decamer with molecular weight of 431 kDa. The optimal pH value of this recombinant protein was 7.5. The divalent ions had no significant effect on Per-catalyzed reaction. The K-m and K-cat/K-m for GDP-4-keto-6-deoxy-D-mannose were 0.09 mM and 2.1 x 10(5) M-1 S-1, and those for L-glutamate were 2 mM and 0.52 x 105 M-1S-1, respectively. Per was used to synthesize GDP-perosamine from GDP-mannose together with recombinant GDP-mannose dehydratase (GMD, E.C. 4.2.1.47). The purified GDP-perosamine was identified by MS and NMR. In summary, this work provided a feasible approach for the synthesis of GDP-perosamine which can lead to the study of LPS biosynthesis of pathogenic E. coli O157 : H7. (C) 2007 Elsevier Inc. All rights reserved.