Shimada T, Tsumura F, Gillam E M, Guengerich F P, Inoue K
Osaka Prefectural Institute of Public Health, 3-69 Nakamichi 1-chome, Higashinari-ku, Osaka, 537-0025, Japan.
Protein Expr Purif. 2000 Oct;20(1):73-80. doi: 10.1006/prep.2000.1302.
Four human cytochrome P450 1B1 (CYP1B1) allelic variants were purified from membranes of Escherichia coli in which respective CYP1B1 cDNAs and human NADPH-P450 reductase cDNA have been introduced. Purified CYP1B1 variants were used to reconstitute 7-ethoxycoumarin O-deethylation activities with purified rabbit liver or recombinant (rat) NADPH-P450 reductase in the phospholipid vesicles and compared with those catalyzed by CYP1B1 enzymes in the membranes of E. coli in monocistronic (by adding the reductase) and bicistronic (without addition of extra reductase) systems. In the bicistronic system, the ratio of expression of NADPH-P450 reductase to CYP1B1 proteins was found to range from 0.2 to 0.5. Purified CYP1B1 enzymes (under optimal reconstitution conditions) catalyzed 7-ethoxycoumarin O-deethylation at rates one-third to one-fourth of those catalyzed by membranes of E. coli coexpressing CYP1B1 and the reductase proteins. Full catalytic activities in reconstituted systems were achieved with a twofold molar excess of NADPH-P450 reductase to CYP1B1; in membranes of E. coli with the monocistronic CYP1B1 construct, an eightfold molar excess of reductase to CYP1B1 was required. However, in membranes of bicistronic constructs, there was no additional stimulation of 7-ethoxycoumarin O-deethylation by extra NADPH-P450 reductase, despite the fact that the molar ratio of expression levels of reductase to CYP1B1 was <0.5. These results suggest that NADPH-P450 reductase produced in the bacterial membranes is more active in interacting with CYP1B1 proteins in the bicistronic system than the reductase added to artificial phospholipid vesicles or bacterial membranes.
从导入了各自的细胞色素P450 1B1（CYP1B1）cDNA和人NADPH-P450还原酶cDNA的大肠杆菌膜中纯化出四种人CYP1B1等位基因变体。纯化后的CYP1B1变体用于在磷脂囊泡中与纯化的兔肝或重组（大鼠）NADPH-P450还原酶一起重建7-乙氧基香豆素O-脱乙基活性，并与单顺反子（通过添加还原酶）和双顺反子（不添加额外还原酶）系统中大肠杆菌膜中的CYP1B1酶催化的活性进行比较。在双顺反子系统中，发现NADPH-P450还原酶与CYP1B1蛋白的表达比例在0.2至0.5之间。纯化的CYP1B1酶（在最佳重建条件下）催化7-乙氧基香豆素O-脱乙基的速率是共表达CYP1B1和还原酶蛋白的大肠杆菌膜催化速率的三分之一至四分之一。当NADPH-P450还原酶与CYP1B1的摩尔过量两倍时，重建系统中可实现完全催化活性；在具有单顺反子CYP1B1构建体的大肠杆菌膜中，还原酶与CYP1B1的摩尔过量需要八倍。然而，在双顺反子构建体的膜中，尽管还原酶与CYP1B1的表达水平摩尔比<0.5，但额外的NADPH-P450还原酶并未对7-乙氧基香豆素O-脱乙基产生额外刺激。这些结果表明，细菌膜中产生的NADPH-P450还原酶在双顺反子系统中与CYP1B1蛋白相互作用时比添加到人工磷脂囊泡或细菌膜中的还原酶更具活性。
