Objectives To use directed evolution to improve YfkO-mediated reduction of the 5-nitroimidazole PET-capable probe SN33623 without impairing conversion of the anti-cancer prodrug CB1954. Results Two iterations of error-prone PCR, purifying selection, and FACS sorting in a DNA damage quantifying GFP reporter strain were used to identify three YfkO variants able to sensitize E. coli host cells to at least 2.4-fold lower concentrations of SN33623 than the native enzyme. Two of these variants were able to be purified in a functional form, and in vitro assays revealed these were twofold and fourfold improved in k cat / K M with SN33623 over wild type YfkO. Serendipitously, the more-active variant was also nearly fourfold improved in k cat / K M versus wild type YfkO in converting CB1954 to a genotoxic drug. Conclusions The enhanced activation of the PET imaging probe SN33623 and CB1954 prodrug exhibited by the lead evolved variant of YfkO offers prospects for improved enzyme-prodrug therapy.

中文翻译：

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枯草芽孢杆菌硝基还原酶 YfkO 的定向进化提高了 PET 探针 SN33623 和 CB1954 前药的激活

目的使用定向进化来改善 YfkO 介导的 5-硝基咪唑 PET 探针 SN33623 的还原，而不损害抗癌前药 CB1954 的转化。结果 使用 DNA 损伤量化 GFP 报告菌株中易错 PCR、纯化选择和 FACS 分选的两次迭代来鉴定三种 YfkO 变体，这些 YfkO 变体能够使大肠杆菌宿主细胞对 SN33623 浓度至少低 2.4 倍。天然酶。这些变体中的两个能够以功能形式被纯化，并且体外测定显示这些变体在使用 SN33623 的 k cat / KM 中比野生型 YfkO 提高了两倍和四倍。意外的是，在将 CB1954 转化为基因毒性药物方面，活性更高的变体在 k cat / KM 方面也比野生型 YfkO 提高了近四倍。