Peyronneau M A, Renaud J P, Truan G, Urban P, Pompon D, Mansuy D
Laboratoire de Chimie et Biochimie Pharmacologiques et Toxicologiques, Centre National de la Recherche Scientifique, Université Paris, France.
Eur J Biochem. 1992 Jul 1;207(1):109-16. doi: 10.1111/j.1432-1033.1992.tb17027.x.
Human liver P450 NF25 (CYP3A4) had been previously expressed in Saccharomyces cerevisiae using the inducible GAL10-CYC1 promoter and the phosphoglycerate kinase gene terminator [Renaud, J. P., Cullin, C., Pompon, D., Beaune, P. and Mansuy, D. (1990) Eur. J. Biochem. 194, 889-896]. The use of an improved expression vector [Urban, P., Cullin, C. and Pompon, D. (1990) Biochimie 72, 463-472] increased the amounts of P450 NF25 produced/culture medium by a factor of five, yielding up to 10 nmol/l. The availability of recently developed host cells that simultaneously overexpress yeast NADPH-P450 reductase and/or express human liver cytochrome b5, obtained through stable integration of the corresponding coding sequences into the yeast genome, led to biotechnological systems with much higher activities of yeast-expressed P450 NF25 and with much better ability to form P450 NF25-iron-metabolite complexes. 9-fold, 8-fold, and 30-fold rate increases were found respectively for nifedipine 1,4-oxidation, lidocaine N-deethylation and testosterone 6 beta-hydroxylation between P450 NF25-containing yeast microsomes from the basic strain and from the strain that both overexpresses yeast NADPH-P450 reductase and expresses human cytochrome b5. Even higher turnovers (15-fold, 20-fold and 50-fold rate increases) were obtained using P450 NF25-containing microsomes from the yeast just overexpressing yeast NADPH-P450 reductase in the presence of externally added, purified rabbit liver cytochrome b5. This is explained by the fact that the latter strain contained the highest level of NADPH-P450 reductase activity. It is noteworthy that for the three tested substrates, the presence of human or rabbit cytochrome b5 always showed a stimulating effect on the catalytic activities and this effect was saturable. Indeed, addition of rabbit cytochrome b5 to microsomes from a strain expressing human cytochrome b5 did not further enhance the catalytic rates. The yeast expression system was also used to study the formation of a P450-NF25-iron-metabolite complex. A P450 Fe(II)-(RNO) complex was obtained upon oxidation of N-hydroxyamphetamine, catalyzed by P450-NF25-containing yeast microsomes. In microsomes from the basic strain expressing P450 NF25, 10% of the starting P450 NF25 was transformed into this metabolite complex, whereas more than 80% of the starting P450 NF25 led to complex formation in microsomes from the strain overexpressing yeast NADPH-P450 reductase.(ABSTRACT TRUNCATED AT 250 WORDS)
人肝P450 NF25（CYP3A4）此前已利用可诱导的GAL10 - CYC1启动子和磷酸甘油酸激酶基因终止子在酿酒酵母中表达[勒诺，J.P.，库兰，C.，蓬庞，D.，博纳，P.和曼叙伊，D.（1990年）《欧洲生物化学杂志》194卷，889 - 896页]。使用一种改进的表达载体[于尔班，P.，库兰，C.和蓬庞，D.（1990年）《生物化学》72卷，463 - 472页]使每升培养基中产生的P450 NF25量增加了五倍，产量高达10纳摩尔。通过将相应编码序列稳定整合到酵母基因组中获得的最近开发的宿主细胞，能够同时过表达酵母NADPH - P450还原酶和/或表达人肝细胞色素b5，这导致生物技术系统中酵母表达的P450 NF25具有更高的活性，且形成P450 NF25 - 铁 - 代谢物复合物的能力更强。在含有P450 NF25的酵母微粒体中，从基础菌株以及同时过表达酵母NADPH - P450还原酶和表达人细胞色素b5的菌株中分别发现硝苯地平1,4 - 氧化、利多卡因N - 脱乙基和睾酮6β - 羟基化的速率分别提高了9倍、8倍和30倍。在外部添加纯化的兔肝细胞色素b5的情况下，使用仅过表达酵母NADPH - P450还原酶的酵母中含有P450 NF25的微粒体，甚至获得了更高的周转率（速率分别提高15倍、20倍和50倍）。这可以用后一种菌株中NADPH - P450还原酶活性水平最高这一事实来解释。值得注意的是，对于三种测试底物，人或兔细胞色素b5的存在总是对催化活性有刺激作用，且这种作用是可饱和的。实际上，向表达人细胞色素b5的菌株的微粒体中添加兔细胞色素b5并不会进一步提高催化速率。酵母表达系统还用于研究P450 - NF25 - 铁 - 代谢物复合物的形成。在含有P450 - NF25的酵母微粒体催化下，N - 羟基苯丙胺氧化后得到了P450 Fe(II) - (RNO)复合物。在表达P450 NF25的基础菌株的微粒体中，起始的P450 NF25有10%转化为这种代谢物复合物，而在过表达酵母NADPH - P450还原酶的菌株的微粒体中，超过80%的起始P450 NF25导致复合物形成。（摘要截选至250字）
