云芝NADPH-细胞色素P450还原酶在大肠杆菌中表达及酶学特性分析

Expression and characterization of NADPH-cytochrome P450 reductase from Trametes versicolor in Escherichia coli

摘要:

云芝Trametes versicolor具有很强的环境有机污染物降解能力，其烟酰胺腺嘌呤二核苷酸-细胞色素P450还原酶 (NADPH-cytochrome P450 reductase，CPR) 为细胞色素P450酶 (Cytochrome P450s，CYPs) 提供电子，参与有机污染物的降解过程。序列分析显示，云芝基因组拥有1个潜在CPR序列和多个潜在CYP序列。为深入研究云芝CPR参与细胞降解有机污染物的分子机制，实验进行了云芝CPR在大肠杆菌中异源表达和酶学特性分析。结果表明，经IPTG诱导后，去除预测的N端膜锚定区域 (氨基酸残基1–24) 的CPR蛋白 (CPRΔ24) 可在重组菌中实现可溶性表达，且表达蛋白的分子量与理论值 (78 kDa) 一致。镍离子亲和层析和分子筛层析纯化后测得其比活性为5.82 U/mg。酶学性质分析显示，重组CPRΔ24的最适温度和pH分别为35 ℃和8.0，并对一些金属离子及有机溶剂具有不同程度的耐受性。酶在35 ℃、pH 8.0反应条件下对NADPH的动力学参数Km和kcat分别为19.7 μmol/L、3.31/s；对底物细胞色素c的动力学参数Km和kcat分别为25.9 μmol/L、10.2/s。以上研究为探究云芝CPR在环境有机污染物降解途径中的功能机制奠定基础。

Abstract:

Trametes versicolor has strong ability to degrade environmental organic pollutants. NADPH-cytochrome P450 reductase (CPR) of T. versicolor transfers electron to cytochrome P450s (CYPs) and participates in the degradation process of organic pollutants. Sequence analysis showed that the genome of T. versicolor contains 1 potential CPR and multiple potential CYP sequences. To further study the molecular mechanism for the involvement of T. versicolor CPR in the cellular degradation of organic pollutants, a CPR gene from T. versicolor was cloned and heterologously expressed in Escherichia coli. Subsequently, the main properties of the recombinant enzyme were investigated. A truncated CPR protein lacking the predicted membrane anchor region (residues 1-24), named CPRΔ24, was overexpressed as a soluble form in E. coli. The recombinant CPRΔ24 protein showed a molecular weight consistent with the theoretical value of 78 kDa. Recombinant CPRΔ24 was purified using a Ni2+-chelating column followed by size exclusion chromatography. The specific activity of the purified CPRΔ24 was 5.82 U/mg. The CPRΔ24 enzyme displayed the maximum activity at 35 ℃ and pH 8.0. It has different degrees of tolerance against several types of metal ions and organic solvents. The apparent Km and kcat values of recombinant CPRΔ24 for NADPH were 19.7 μmol/L and 3.31/s, respectively, and those for the substrate cytochrome c were 25.9 μmol/L and 10.2/s, respectively, under conditions of 35 ℃ and pH 8.0. The above research provides the basis for exploring the functional mechanism of T. versicolor CPR in the degradation pathway of environmental organic pollutants.