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

Welin, Martin; Skovgaard, T.; Knecht, W.; Zhu, C.; Berenstein, D.; Munch-Petersen, B.; Piskur, Jure LU and Eklund, Hans (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)
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author
organization
publishing date
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
Federation of European Neuroscience Societies and Blackwell Publishing Ltd
external identifiers
  • wos:000230293100022
  • scopus:22544476496
ISSN
1742-464X
DOI
10.1111/j.1742-4658.2005.04803.x
language
English
LU publication?
yes
id
b1131ef2-25a4-4ff6-99a5-4a9b93aa66d9 (old id 744978)
date added to LUP
2008-01-09 15:08:22
date last changed
2017-06-18 03:46:24
@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},
  keyword      = {deoxyribonucleoside kinase,feed-back inhibition,fruit fly,structure-function},
  language     = {eng},
  number       = {14},
  pages        = {3733--3742},
  publisher    = {Federation of European Neuroscience Societies and Blackwell Publishing Ltd},
  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},
  volume       = {272},
  year         = {2005},
}