Gillam E M, Guo Z, Ueng Y F, Yamazaki H, Cock I, Reilly P E, Hooper W D, Guengerich F P
Department of Physiology, University of Queensland, St. Lucia, Australia.
Arch Biochem Biophys. 1995 Mar 10;317(2):374-84. doi: 10.1006/abbi.1995.1177.
Cytochrome P450 (P450) 3A5 is a human enzyme with 85% amino acid sequence identity to the more predominantly expressed P450 3A4 and has been reported to have overlapping catalytic specificity. The 5'-terminus of a P450 3A5 cDNA was modified for optimal expression in Escherichia coli using the vector pCW, by aligning the MALLLAVFL N-terminal sequence of recombinant bovine P450 17A (H. J. Barnes, M. P. Arlotto, and M. R. Waterman, (1991) Proc. Natl. Acad. Sci. USA 88, 5597-5601) to the 3A5 cDNA. Two constructs were made, differing by their identity with the modified 3A4 N-terminal sequence (E. M. J. Gillam, T. Baba, B-R. Kim, S. Ohmori, and F. P. Guengerich, (1993) Arch. Biochem. Biophys. 305, 123-131). The first modified sequence (3A5#1) was identical to recombinant P450 3A4 up to codon 15, the 3A5 sequence being introduced thereafter. In the other (3A5#2), the successful 3A4 N-terminal nucleotide sequence was attached to codon 30. The yield was greater than fourfold higher in the first construct [up to 260 nmol (liter culture)-1]. The recombinant P450 3A5 (construct 1) was purified to electrophoretic homogeneity using a variation of a three-step procedure developed previously for P450 3A4, with an overall yield of approximately 40%. Purified P450 3A5 was active in nifedipine oxidation, testosterone 6 beta-hydroxylation, aflatoxin 3 alpha-hydroxylation and 8,9-epoxidation, ethylmorphine N-demethylation, erythromycin N-demethylation, and d-benzphetamine N-demethylation. The reconstitution of nifedipine oxidation, testosterone 6 beta-hydroxylation, and the aflatoxin oxidation activities showed dependence upon the presence of cytochrome b5, divalent cations, phospholipid mixtures, glutathione, and cholate similar to that previously found for purified P450 3A4. However, rates of the N-demethylations of ethylmorphine, erythromycin, and d-benzphetamine were as high or higher for P450 3A5 than P450 3A4 and were not particularly dependent upon modifications of reconstitution systems [corrected].
细胞色素P450（P450）3A5是一种人类酶，其氨基酸序列与表达更为广泛的P450 3A4有85%的同一性，据报道具有重叠的催化特异性。使用载体pCW对P450 3A5 cDNA的5'-末端进行了修饰，以便在大肠杆菌中实现最佳表达，方法是将重组牛P450 17A的MALLLAVFL N-末端序列（H. J. 巴恩斯、M. P. 阿洛托和M. R. 沃特曼，（1991年）《美国国家科学院院刊》88，5597 - 5601）与3A5 cDNA进行比对。构建了两种构建体，它们与修饰后的3A4 N-末端序列的一致性有所不同（E. M. J. 吉勒姆、T. 巴巴、B-R. 金、S. 大森和F. P. 根格里奇，（1993年）《生物化学与生物物理学报》305，123 - 131）。第一个修饰序列（3A5#1）与重组P450 3A4在密码子15之前相同，此后引入3A5序列。在另一个构建体（3A5#2）中，成功的3A4 N-末端核苷酸序列连接到密码子30。在第一个构建体中产量提高了四倍多[高达260 nmol（升培养物）-1]。使用先前为P450 3A4开发的三步法的一种变体，将重组P450 3A5（构建体1）纯化至电泳纯，总产率约为40%。纯化的P450 3A5在硝苯地平氧化、睾酮6β-羟基化、黄曲霉毒素3α-羟基化和8,9-环氧化、乙基吗啡N-去甲基化、红霉素N-去甲基化以及d-苄非他明N-去甲基化反应中具有活性。硝苯地平氧化、睾酮6β-羟基化和黄曲霉毒素氧化活性的重组显示，其依赖于细胞色素b5、二价阳离子、磷脂混合物、谷胱甘肽和胆酸盐的存在，这与先前对纯化的P450 3A4的发现相似。然而，P450 3A5对乙基吗啡、红霉素和d-苄非他明的N-去甲基化速率与P450 3A4相同或更高，并且对重组系统的修饰没有特别的依赖性[已校正]。
