tailieunhanh - Báo cáo khoa học: Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D

TheDrosophila melanogasterdeoxyribonucleoside 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 activ-ity with 3¢-modified nucleoside analogs like 3¢-azidothymidine (AZT) was nearly unchanged. | ềFEBS Journal Structural basis for the changed substrate specificity of Drosophila melanogaster deoxyribonucleoside kinase mutant N64D Martin Welin1 Tine Skovgaard2 Wolfgang Knecht3 Chunying Zhu2 Dvora Berenstein2 Birgitte Munch-Petersen2 Jure PiSkur3 t and Hans Eklund1 1 Department of Molecular Biology Swedish University of AgriculturalSciences BiomedicalCenter Uppsala Sweden 2 Department of Life Sciences and Chemistry Roskilde University Denmark 3 BioCentrum-DTU TechnicalUniversity of Denmark Lyngby Denmark Keywords crystal structure feedback inhibition gene therapy pro-drug activation Correspondence H. Eklund Department of Molecular Biology Swedish University of Agricultural Sciences Box 590 BiomedicalCenter S-751 24 Uppsala Sweden Fax 46 18 53 69 71 Tel 46 18 475 4559 E-mail hasse@ Present address AstraZeneca R D Molndal Sweden tPresent address Celland Organism Biology Lund University Sweden Received 12 April2005 revised 30 May 2005 accepted 3 June 2005 doi 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 .