Liu Lingfeng, Li Yongfeng, Liotta Dennis, Lutz Stefan
Department of Chemistry, Emory University, Atlanta, GA 30322, USA.
Nucleic Acids Res. 2009 Jul;37(13):4472-81. doi: 10.1093/nar/gkp400. Epub 2009 May 27.
Nucleoside analogs (NAs) represent an important category of prodrugs for the treatment of viral infections and cancer, yet the biological potency of many analogs is compromised by their inefficient activation through cellular 2'-deoxyribonucleoside kinases (dNKs). We herein report the directed evolution and characterization of an orthogonal NA kinase for 3'-deoxythymidine (ddT), using a new FACS-based screening protocol in combination with a fluorescent analog of ddT. Four rounds of random mutagenesis and DNA shuffling of Drosophila melanogaster 2'-deoxynucleoside kinase, followed by FACS analysis, yielded an orthogonal ddT kinase with a 6-fold higher activity for the NA and a 20-fold k(cat)/K(M) preference for ddT over thymidine, an overall 10,000-fold change in substrate specificity. The contributions of individual amino acid substitutions in the ddT kinase were evaluated by reverse engineering, enabling a detailed structure-function analysis to rationalize the observed changes in performance. Based on our results, kinase engineering with fluorescent NAs and FACS should prove a highly versatile method for evolving selective kinase:NA pairs and for studying fundamental aspects of the structure-function relationship in dNKs.
核苷类似物（NAs）是用于治疗病毒感染和癌症的一类重要前药，然而许多类似物的生物学效力因通过细胞2'-脱氧核糖核苷激酶（dNKs）的低效激活而受到影响。我们在此报告了一种针对3'-脱氧胸苷（ddT）的正交NA激酶的定向进化和表征，使用了一种基于荧光激活细胞分选（FACS）的新筛选方案，并结合了ddT的荧光类似物。对果蝇2'-脱氧核苷激酶进行四轮随机诱变和DNA改组，随后进行FACS分析，得到了一种正交ddT激酶，其对该核苷类似物的活性提高了6倍，对ddT的k(cat)/K(M)偏好性比对胸苷高20倍，底物特异性总体变化了10000倍。通过逆向工程评估了ddT激酶中单个氨基酸取代的贡献，从而能够进行详细的结构-功能分析，以合理化观察到的性能变化。基于我们的结果，利用荧光核苷类似物和FACS进行激酶工程应是一种高度通用的方法，可用于进化选择性激酶：核苷类似物对，并用于研究dNKs中结构-功能关系的基本方面。
