School of Chemistry and Molecular Biosciences, The University of Queensland, St. Lucia, Brisbane 4072, Australia.
Chem Res Toxicol. 2012 Sep 17;25(9):1964-74. doi: 10.1021/tx300281g. Epub 2012 Aug 29.
Cytochrome P450 2F1 (P450 2F1) is expressed exclusively in the human respiratory tract and is implicated in 3-methylindole (3MI)-induced pneumotoxicity via dehydrogenation of 3MI to a reactive electrophilic intermediate, 3-methyleneindolenine (3-MEI). Studies of P450 2F1 to date have been limited by the failure to express this enzyme in Escherichia coli. By contrast, P450 2F3, a caprine homologue that shares 84% sequence identity with P450 2F1 (86 amino acid differences), has been expressed in E. coli at yields greater than 250 nmol/L culture. We hypothesized that a limited number of sequence differences between P450s 2F1 and 2F3 could limit P450 2F1 expression in E. coli and that problematic P450 2F1 sequence elements could be identified by directed evolution. A library of P450 2F1/2F3 mutants was created by DNA family shuffling and screened for expression in E. coli. Three generations of DNA shuffling revealed a mutant (named JH_2F_F3_1_007) with 96.5% nucleotide sequence identity to P450 2F1 and which expressed 119 ± 40 pmol (n = 3, mean ± SD) hemoprotein in 1 mL microaerobic cultures. Across all three generations, two regions were observed where P450 2F3-derived sequence was consistently substituted for P450 2F1 sequence in expressing mutants, encoding nine amino acid differences between P450s 2F1 and 2F3: nucleotides 191-278 (amino acids 65-92) and 794-924 (amino acids 265-305). Chimeras constructed to specifically test the importance of these two regions confirmed that P450 2F3 sequence is essential in both regions for expression in E. coli but that other non-P450 2F1 sequence elements outside of these regions also improved the expression of mutant JH_2F_F3_1_007. Mutant JH_2F_F3_1_007 catalyzed the dehydrogenation of 3MI to 3-MEI as indicated by the observation of glutathione adducts after incubation in the presence of glutathione. The JH_2F_F3_1_007 protein differs from P450 2F1 at only 20 amino acids and should facilitate further studies of the structure-activity relationships of P450s of the 2F subfamily.
细胞色素 P450 2F1（P450 2F1）仅在人体呼吸道中表达，通过将 3-甲基吲哚（3MI）脱氢为反应性亲电中间产物 3-亚甲基吲哚啉（3-MEI），参与 3MI 诱导的肺毒性。迄今为止，对 P450 2F1 的研究由于未能在大肠杆菌中表达这种酶而受到限制。相比之下，山羊同源物 P450 2F3 已在大肠杆菌中以超过 250nmol/L 培养物的产率表达，其与 P450 2F1 的序列同一性为 84%（86 个氨基酸差异）。我们假设 P450s 2F1 和 2F3 之间有限数量的序列差异可能限制了 P450 2F1 在大肠杆菌中的表达，并且可以通过定向进化来鉴定有问题的 P450 2F1 序列元件。通过 DNA 家族改组创建了 P450 2F1/2F3 突变体文库，并在大肠杆菌中进行了表达筛选。经过三代 DNA 改组，发现了一个突变体（命名为 JH_2F_F3_1_007），其核苷酸序列与 P450 2F1 有 96.5%的同一性，在微需氧培养物 1mL 中表达 119±40pmol（n=3，平均值±标准差）血红素蛋白。在所有三代中，观察到两个区域，其中 P450 2F3 衍生的序列始终替代表达突变体中的 P450 2F1 序列，在 P450s 2F1 和 2F3 之间编码九个氨基酸差异：核苷酸 191-278（氨基酸 65-92）和 794-924（氨基酸 265-305）。专门用于测试这两个区域重要性的嵌合体证实，P450 2F3 序列对于大肠杆菌中的表达在这两个区域都是必不可少的，但这两个区域之外的其他非 P450 2F1 序列元件也提高了突变体 JH_2F_F3_1_007 的表达。突变体 JH_2F_F3_1_007 催化 3MI 脱氢为 3-MEI，这可以通过在存在谷胱甘肽时孵育后观察到谷胱甘肽加合物来证实。JH_2F_F3_1_007 蛋白与 P450 2F1 仅在 20 个氨基酸处不同，应有助于进一步研究 2F 亚家族 P450 的结构-活性关系。
