Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D
(2005) In The FEBS Journal 272(14). p.3733-3742- 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... (More)
- 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. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/744978
- author
- Welin, Martin ; Skovgaard, T. ; Knecht, W. LU ; Zhu, C. ; Berenstein, D. ; Munch-Petersen, B. ; Piskur, Jure LU and Eklund, Hans
- publishing date
- 2005
- type
- Contribution to journal
- publication status
- published
- subject
- keywords
- deoxyribonucleoside kinase, feed-back inhibition, fruit fly, structure-function
- in
- The FEBS Journal
- volume
- 272
- issue
- 14
- pages
- 3733 - 3742
- publisher
- Wiley-Blackwell
- external identifiers
-
- wos:000230293100022
- scopus:22544476496
- ISSN
- 1742-464X
- DOI
- 10.1111/j.1742-4658.2005.04803.x
- language
- English
- LU publication?
- no
- id
- b1131ef2-25a4-4ff6-99a5-4a9b93aa66d9 (old id 744978)
- date added to LUP
- 2016-04-01 12:25:53
- date last changed
- 2022-01-27 03:38:32
@article{b1131ef2-25a4-4ff6-99a5-4a9b93aa66d9, 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.}}, author = {{Welin, Martin and Skovgaard, T. and Knecht, W. and Zhu, C. and Berenstein, D. and Munch-Petersen, B. and Piskur, Jure and Eklund, Hans}}, issn = {{1742-464X}}, keywords = {{deoxyribonucleoside kinase; feed-back inhibition; fruit fly; structure-function}}, language = {{eng}}, number = {{14}}, pages = {{3733--3742}}, publisher = {{Wiley-Blackwell}}, series = {{The FEBS Journal}}, title = {{Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D}}, url = {{http://dx.doi.org/10.1111/j.1742-4658.2005.04803.x}}, doi = {{10.1111/j.1742-4658.2005.04803.x}}, volume = {{272}}, year = {{2005}}, }