Sabini Elisabetta, Ort Stephan, Monnerjahn Christian, Konrad Manfred, Lavie Arnon
University of Illinois at Chicago, Department of Biochemistry and Molecular Biology, 1819 West Polk Street, Chicago, Illinois 60612, USA.
Nat Struct Biol. 2003 Jul;10(7):513-9. doi: 10.1038/nsb942.
Human deoxycytidine kinase (dCK) phosphorylates the natural deoxyribonucleosides deoxycytidine (dC), deoxyguanosine (dG) and deoxyadenosine (dA) and is an essential enzyme for the phosphorylation of numerous nucleoside analog prodrugs routinely used in cancer and antiviral chemotherapy. For many of these compounds, the phosphorylation step catalyzed by dCK is the rate-limiting step in their overall activation pathway. To determine the factors that limit the phosphorylation efficiency of the prodrug, we solved the crystal structure of dCK to a resolution of 1.6 A in complex with its physiological substrate deoxycytidine and with the prodrugs AraC and gemcitabine. The structures reveal the determinants of dCK substrate specificity. Especially relevant to new prodrug development is the interaction between Arg128 and the hydrogen-bond acceptor at the sugar 2'-arabinosyl position of AraC and gemcitabine. On the basis of the structures, we designed a catalytically superior dCK variant that could be used in suicide gene-therapy applications.
人类脱氧胞苷激酶（dCK）可将天然脱氧核糖核苷脱氧胞苷（dC）、脱氧鸟苷（dG）和脱氧腺苷（dA）磷酸化，是癌症和抗病毒化疗中常用的多种核苷类似物前药磷酸化过程所必需的一种酶。对于其中许多化合物而言，由dCK催化的磷酸化步骤是其整体激活途径中的限速步骤。为了确定限制前药磷酸化效率的因素，我们解析了dCK与它的生理底物脱氧胞苷以及前药阿糖胞苷（AraC）和吉西他滨形成复合物的晶体结构，分辨率达到1.6埃。这些结构揭示了dCK底物特异性的决定因素。与新前药开发特别相关的是精氨酸128与阿糖胞苷和吉西他滨糖基2'-阿拉伯糖基位置上的氢键受体之间的相互作用。基于这些结构，我们设计了一种催化性能更优的dCK变体，可用于自杀基因治疗应用。
