Heterologous expression and characterization of soluble recombinant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Actinosynnema pretiosum ssp auranticum ATCC31565 through co-expression with Chaperones in Escherichia coli
Heterologous expression and characterization of soluble recombinant 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Actinosynnema pretiosum ssp auranticum ATCC31565 through co-expression with Chaperones in Escherichia coli
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DOI：
10.1016/j.pep.2012.01.013
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发表时间：
2012-04-01
影响因子：
1.6
通讯作者：
Hua, Qiang
中科院分区：
文献类型：
--
作者：
Ma, Na;Wei, Liujing;Hua, Qiang
3-脱氧- d -阿拉伯-庚糖酸-7-磷酸合成酶（DAHPS） （EC 2.5.1.54）催化莽草酸途径的第一步，是植物和微生物中芳香族化合物的生物合成途径。在放线酸综合征中，aroF基因（GenBank: AF056968.1）编码DAHPS凝聚磷酸烯醇丙酮酸（PEP）和d - 4-磷酸红酶（E4P）生成DAHP。本研究构建了重组pET28a-aroF质粒，通过与伴侣蛋白GroEL/GroES共表达，成功地在大肠杆菌中以可溶性形式表达了a . pretiosum DAHPS。对表达蛋白的纯化和动力学特性进行了研究。结果表明，该菌株对底物PEP有明显的抑制作用，但对E4P不敏感。纯化后的酶被EDTA完全失活，但被几种二价金属离子，特别是Mn2+和Co2+强激活。(C) 2012爱思唯尔公司版权所有。
3-Deoxy-D-arabino-heptulosonate-7-phosphate synthase (DAHPS), (EC 2.5.1.54) catalyzes the first step of the shikimate pathway, the route for the biosynthesis of aromatic compounds in plants and microbes. In Actinosynnema pretiosum, the aroF gene (GenBank: AF056968.1) encodes DAHPS to condensate phosphoenolpyruvate (PEP) and D-erythrose 4-phosphate (E4P) to generate DAHP. In this study, a recombinant pET28a-aroF plasmid was constructed and A. pretiosum DAHPS was successfully expressed in soluble form by co-expression with chaperonins GroEL/GroES in Escherichia coli. The purification and kinetic characterization of the expressed protein were then investigated. The DAHPS originated from A. pretiosum demonstrated a pronounced substrate inhibition by PEP but was not sensitive to E4P. The purified enzyme was completely inactivated by EDTA but potently activated by several bivalent metal ions, especially Mn2+ and Co2+. (C) 2012 Elsevier Inc. All rights reserved.