Functional characterization of Class II 5-enopyruvylshikimate-3-phosphate synthase from Halothermothrix orenii H168 in Escherichia coli and transgenic Arabidopsis

文献类型： 外文期刊

第一作者： Tian, Yong-Sheng

作者： Tian, Yong-Sheng;Xu, Jing;Xiong, Ai-Sheng;Zhao, Wei;Gao, Feng;Fu, Xiao-Yan;Peng, Ri-He;Yao, Quan-Hong

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关键词： 5-Enopyruvylshikimate-3-phosphate synthase;Enzyme kinetic values;Halothermothrix orenii;Transgenic Arabidopsis

期刊名称：APPLIED MICROBIOLOGY AND BIOTECHNOLOGY （ 影响因子：4.813； 五年影响因子：4.697 ）

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收录情况： SCI

摘要： Although a large number of AroA enzymes (5-enopyruvylshikimate-3-phosphate synthase [EPSPS]) have been identified, cloned and tested for glyphosate resistance, only AroA variants derived from Agrobacterium tumefaciens strain CP4 have been successfully used commercially. We have now used a polymerase chain reaction (PCR)-based two-step DNA synthesis (PTDS) method to synthesize an aroA gene (aroA _(H. orenii)) from Halothermothrix orenii H168 encoding a new EPSPS similar to AroA _(A. tumefaciens CP4). AroA _(H. orenii) was then expressed in Escherichia coli and key kinetic values of the purified enzyme were determined. Kinetic analysis of AroA _(H. orenii) indicated that the full-length enzyme exhibited increased tolerance to glyphosate compared with E. coli AroA _(E. coli) while retaining a high affinity for the substrate phosphoenolpyruvate. Transgenic Arabidopsis plants containing aroA H. orenii were resistant to 15 mM glyphosate. Site-directed mutagenesis showed that residues Thr355Ser affected the affinity of AroA _(H. orenii) for glyphosate, providing further evidence that specific amino acid residues are responsible for differences in enzymatic behavior among different AroA enzymes.

分类号： Q939.9

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