Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D

M. Welin, T. Skovgaard, Wolfgang Knecht, C.Y. Zhu, D. Berenstein, B. Munch-Petersen, Jure Piskur, H. Eklund

    Research output: Contribution to journalJournal articleResearchpeer-review

    Abstract

    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.
    Original languageEnglish
    JournalFEBS Journal
    Volume272
    Issue number14
    Pages (from-to)3733-3742
    ISSN1742-464X
    Publication statusPublished - 2005

    Keywords

    • gene therapy
    • feedback inhibition
    • pro-drug activation
    • crystal structure

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