Functional expression and purification of CYP93C20, a plant membrane- associated cytochrome P450 from Medicago truncatula
Functional expression and purification of CYP93C20, a plant membrane- associated cytochrome P450 from Medicago truncatula
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CYP93C20（一种来自蒺藜苜蓿的植物膜相关细胞色素 P450）的功能表达和纯化
DOI：
10.1016/j.pep.2018.04.017
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发表时间：
2018-10-01
影响因子：
1.6
通讯作者：
Hu, Hongli
中科院分区：
文献类型：
--
作者：
Chang, Zhenzhan;Wang, Xiaoqiang;Hu, Hongli
植物拥有大量生物合成细胞色素 P450 酶。尽管这些酶对于生物活性植物次生代谢物的合成很重要，但迄今为止仅获得了两种植物 P450 结构。异黄酮合酶 (IFS) 是一种膜相关细胞色素 P450 酶，催化异黄酮生物合成的入口点反应。来自模型豆科植物蒺藜苜蓿 (CYP93C20) 的 IFS 是通过删除跨膜结构域并在 N 末端插入亲水性多肽和在 C 末端插入四个组氨酸标签而设计的。截短的形式表现出显着增强的表达和溶解度。工程酶在大肠杆菌XL1-blue细胞中表达，并通过Ni2+-NTA亲和层析和尺寸排阻层析进行纯化。通过酶测定、还原一氧化碳差值光谱和肽质量指纹图谱对纯化的酶进行表征。工程可溶性酶表现出与酵母中表达的全长膜相关酶相同的活性。这些研究提出了一种工程化植物膜相关 P450 的方法，以增强表达和溶解度，用于机械和结构研究。
Plants possess very large numbers of biosynthetic cytochrome P450 enzymes. In spite of the importance of these enzymes for the synthesis of bioactive plant secondary metabolites, only two plant P450 structures has been obtained to date. Isoflavone synthase (IFS) is a membrane-associated cytochrome P450 enzyme catalyzing the entry-point reaction into isoflavonoid biosynthesis. IFS from the model legume Medicago truncatula (CYP93C20) was engineered by deleting the membrane-spanning domain and inserting a hydrophilic polypeptide in the N-terminus and a four histidine tag at the C-terminus. The truncated form exhibited dramatically enhanced expression and solubility. The engineered enzyme was expressed in Escherichia coil XL1-blue cells and was purified by Ni2+-NTA affinity chromatograph and size-exclusion chromatograph. The purified enzyme was characterized by enzyme assay, reduced carbon monoxide difference spectroscopy and peptide mass fingerprinting. The engineered soluble enzyme exhibited the same activity as the full length membrane-associated enzyme expressed in yeast. These studies suggest an approach for engineering plant membrane-associated P450s with enhanced expression and solubility for mechanistic and structural studies.