5种人细胞色素P450 2A13突变体的异源表达及其香豆素代谢活性

Heterogenous expression of five human cytochrome P450 2A13 mutants and their catalytic activities of coumarin

目的重组表达人细胞色素P450（CYP）2A13突变体CYP2A13＊2,CYP2A13＊5,CYP2A13＊6,CYP2A13＊8和CYP2A13＊9,并研究CYP2A13突变体与野生型CYP2A13（CYP2A13＊1）对香豆素的代谢活性上的差异。方法通过点突变法构建各CYP2A13突变体。用杆状病毒表达系统制备含各突变体c DNA的重组病毒,并将其与含人源还原型烟酰胺腺嘌呤二核苷磷酸P450氧化还原酶（POR,野生型）的重组杆状病毒共转染昆虫草地夜蛾细胞（sf9）,共表达CYP2A13突变体和POR重组蛋白。将表达产物制备成微粒体与香豆素（0.25~25μmol·L-1）体外孵育30 min,高效液相色谱法测定各重组酶对香豆素的代谢活性。结果Western蛋白印迹结果表明,各重组酶在sf9细胞中得到了表达。体外代谢实验显示,CYP2A13野生型及其突变体蛋白代谢香豆素在Km值上无显著差异,而在Vmax（mol·min-1·mol-1CYP）上,CYP2A13＊2（0.58±0.05,P〈0.05）、CYP2A13＊5（0.71±0.08,P〈0.01）、CYP2A13＊6（0.84±0.09,P〈0.01）、CYP2A13＊9（0.58±0.04,P〈0.05）等突变体与CYP2A13＊1（0.33±0.06）相比有显著性差异,使得这4个突变体的代谢效率增加了40%~108%。结论体外成功表达了具有活性的CYP2A13＊2,CYP2A13＊5,CYP2A13＊6,CYP2A13＊8和CYP2A13＊9重组蛋白。CYP2A13＊2,CYP2A13＊5,CYP2A13＊6和CYP2A13＊9等突变体的香豆素代谢活性明显高于CYP2A13＊1。

OBJECTIVE To construct and express recombinant human P450 2A13 mutants CYP2A13＊2, CYP2A13＊5, CYP2A13＊6, CYP2A13＊8 and CYP2A13＊9, and to determine the function- al consequences, if any, that CYP2A13 mutants might have on CYP2A13-catalysed coumarin metabo- lism. METHODS The wild-type CYP2A13 gene was used as template to obtain CYP2A13 mutants by site-directed mutation PCR. Bac-to-Bac Baculovirus expression system was employed to produce re- combinant baculovirus carrying cDNAs of CYP2A13 mutants. Spodoptera frugiperc/a 9 （Sf9） cells were co-infected with recombinant baculovirus of CYP2A13 mutants and human NADPH-P450 oxidoreduc- tase （POR, wildtype） to express CYP2A13 proteins. The microsome containing recombinant proteins was prepared and incubated with coumarin （0.25-25 μmol·L^-1） for 30 min. The coumarin 7-hydroxyl- ation activities of CYP2A13 mutants were determined by HPLC. RESULTS A single band of 56 ku was detected in the microsomes prepared from the Sf9 cells transfected with bacmids containing CYP2A13 cDNAs. CYP2A13 mutants and CYP2A13＊1 had similar K,,values. However, the Vr~ values （mol- min-1. mo1-1 CYP） of CYP2A13＊2 （0.58±0.05, P〈0.05）, CYP2A13＊5 （0.71±0.08, P〈0.01）, CYP2A13＊6 （0.84± 0.09, P〈 0.01） and CYP2A13＊9 （0.58 ±0.04, P〈 0.05） were significantly higher those of CYP2A13＊1 （0.33±0.06）, which resulted in a 40%- 108% increase of catalytic efficiency. CONCLUSION CYP2A13＊2, CYP2A13＊5, CYP2A13＊6, CYP2A13＊8 and CYP2A13＊9 with metabolic activities are successfully expressed using Bac-to-Bac baculovirus expression system. The results of coumarin metabolism show that the coumarin 7-hydroxylation activities of CYP2A13＊2, CYP2A13＊5, CYP2A13＊6 and CYP2A13＊9 are significantly higher than those of CYP2A13＊1.