Characterization of native and histidine-tagged deoxyxylulose 5-phosphate reductoisomerase from the cyanobacterium Synechocystis sp. PCC6803.
Characterization of native and histidine-tagged deoxyxylulose 5-phosphate reductoisomerase from the cyanobacterium Synechocystis sp. PCC6803.
复制标题
蓝藻集胞藻属天然和组氨酸标记的脱氧木酮糖 5-磷酸还原异构酶的表征。
DOI：
10.1016/j.bbapap.2003.08.005
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发表时间：
2003
期刊：
影响因子：
--
通讯作者：
Proteau,PhilipJ
中科院分区：
文献类型：
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作者：
Yin,Xihou;Proteau,PhilipJ
蓝藻聚球藻DXR基因编码1-脱氧-D-木糖5-磷酸还原异构酶(DXR)。在大肠杆菌中表达了PCC6803，可产生天然和N末端组氨酸标记形式的DXR。用阴离子交换层析或金属亲和层析和凝胶过滤从细胞提取液中纯化酶。纯化的重组天然酶和组氨酸标记的酶在十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)上均显示一条单一条带，分别对应于计算的亚基相对分子质量为42,500和46,700。经天然PAGE分析，两种酶在还原条件下均为二聚体。对酶的动力学性质进行了表征，仅观察到微小的变化，表明N-末端组氨酸标签对酶活性影响不大。这两种酶与以前从其他来源鉴定的还原异构酶具有相似的性质。首次测定了天然DXR中金属离子Mn2+、Mg2+和Co2+的Km值，其中Mg2+的Km值约为Mn2+和Co2+的Km值的200倍。
The dxr gene encoding the 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) from the cyanobacterium Synechocystis sp. PCC6803 was expressed in Escherichia coli to produce both the native and N-terminal histidine-tagged forms of DXR. The enzymes were purified from the cell extracts using either anion exchange chromatography or metal affinity chromatography and gel filtration. The purified recombinant native and histidine-tagged enzymes each displayed a single band on sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) gels, corresponding to the calculated subunit molecular weights of 42,500 and 46,700, respectively. By native PAGE, both enzymes were dimers under reducing conditions. The kinetic properties for the enzymes were characterized and only minor variations were observed, demonstrating that the N-terminal histidine tag does not greatly affect the activity of the enzyme. Both enzymes had similar properties to previously characterized reductoisomerases from other sources. The Km's for the metal ions Mn2+, Mg2+, and Co2+were determined for native DXR for the first time, with the Kmfor Mg2+being approximately 200-fold higher than the Km's for Mn2+and Co2+.