Welin Martin, Skovgaard Tine, Knecht Wolfgang, Zhu Chunying, Berenstein Dvora, Munch-Petersen Birgitte, Piskur Jure, Eklund Hans
Department of Molecular Biology, Swedish University of Agricultural Sciences, Biomedical Center, Uppsala, Sweden.
FEBS J. 2005 Jul;272(14):3733-42. doi: 10.1111/j.1742-4658.2005.04803.x.
The Drosophila melanogaster deoxyribonucleoside kinase (Dm-dNK) double mutant N45D/N64D was identified during a previous directed evolution study. This mutant enzyme had a decreased activity towards the natural substrates and decreased feedback inhibition with dTTP, whereas the activity with 3'-modified nucleoside analogs like 3'-azidothymidine (AZT) was nearly unchanged. Here, we identify the mutation N64D as being responsible for these changes. Furthermore, we crystallized the mutant enzyme in the presence of one of its substrates, thymidine, and the feedback inhibitor, dTTP. The introduction of the charged Asp residue appears to destabilize the LID region (residues 167-176) of the enzyme by electrostatic repulsion and no hydrogen bond to the 3'-OH is made in the substrate complex by Glu172 of the LID region. This provides a binding space for more bulky 3'-substituents like the azido group in AZT but influences negatively the interactions between Dm-dNK, substrates and feedback inhibitors based on deoxyribose. The detailed picture of the structure-function relationship provides an improved background for future development of novel mutant suicide genes for Dm-dNK-mediated gene therapy.
果蝇脱氧核糖核苷激酶（Dm-dNK）双突变体N45D/N64D是在之前的定向进化研究中鉴定出来的。该突变酶对天然底物的活性降低，对dTTP的反馈抑制作用减弱，而对3'-修饰的核苷类似物如3'-叠氮胸苷（AZT）的活性几乎不变。在此，我们确定突变N64D是造成这些变化的原因。此外，我们在其一种底物胸苷和反馈抑制剂dTTP存在的情况下使突变酶结晶。带电荷的天冬氨酸残基的引入似乎通过静电排斥使酶的LID区域（第167 - 176位氨基酸残基）不稳定，并且LID区域的Glu172在底物复合物中未与3'-OH形成氢键。这为像AZT中的叠氮基这样更大体积的3'-取代基提供了一个结合空间，但对基于脱氧核糖的Dm-dNK、底物和反馈抑制剂之间的相互作用产生负面影响。结构 - 功能关系的详细情况为未来开发用于Dm-dNK介导的基因治疗的新型突变自杀基因提供了更好的背景。
