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

Abstract

Deoxyribonucleoside kinases (dNKs; EC 2.7.1.145) catalyze the initial, and usually rate-determining step in the synthesis of the four DNA precursors (dNTPs) through the salvage pathway. These enzymes transfer the c-phosphoryl group from ATP to deoxyribonucleosides (dN) and form the corresponding dNMPs [1]. In the cell, dNMPs are quickly phosphorylated to dNDPs and dNTPs by ubiquitous mono-and diphosphate deoxyribonucleoside kinases.Deoxyribonucleoside kinases are also responsible for activation (initial phosphorylation) of nontoxic nucleoside analogs such as azidothymidine (AZT) and acyclovir (ACV) used in the treatment of cancer and viral diseases. After further phosphorylation by other cellular kinases the triphosphorylated nucleoside analogs are incorporated into DNA and cause chain termination and cell death [2]. Alternatively, they inhibit the DNA synthesizing machinery or initiate apoptosis [3]. 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.

Drosophila deoxyribonucleoside kinase mutants with enhanced ability to phosphorylate purine analogs

“…Factors that could play a role here would be differences in the feedback inhibition and the relative half-life of the mutant Dm-dNKs in E. coli, as well as the NA uptake, its catabolism and the further phosphorylation to the di-and triphosphate level. Differences in the feedback regulation between wild-type Dm-dNK and mutant Dm-dNK have been described previously, 6,25,26 resulting mainly in a loss of inhibition by thymidine triphosphate (dTTP). In fact it has been shown that some mutant Dm-dNK instead can use dTTP as phosphate donor.…”

“…Recently, the N64D mutation has been determined to be crucial for the enhanced toxicity of AZT mediated by the N45D/N64D Dm-dNK double mutant, and the structure of the N64D mutant of Dm-dNK has been solved and its implication discussed in detail. 26 This mutant has been first recognized for its ability to enhance the cytotoxicity for pyrimidine analogs especially AZT by providing space for its bulky 3 0 -azide group. This was also displayed in our study by B10 when tested in E. coli (Table 1).…”

Structure-guided Engineering of Human Thymidine Kinase 2 as a Positron Emission Tomography Reporter Gene for Enhanced Phosphorylation of Non-natural Thymidine Analog Reporter Probe

“…To reduce the susceptibility of TK2 to dTTP-mediated feedback inhibition, we took advantage of the 40% sequence identity between human TK2 and Drosophila melanogaster deoxyribonucleoside kinase (Dm-DNK) (53) and of the identification of a point mutation (N64D) in Dm-DNK that has been shown to reduce the effect of dTTP feedback inhibition (54). The residue in TK2 corresponding to Asn-64 in D. melanogaster deoxyribonucleoside kinase is Asn-93; the corresponding mutation in TK2 is N93D.…”

Enzymes To Die For: Exploiting Nucleotide Metabolizing Enzymes for Cancer Gene Therapy

“…Results suggest the N64D mutation is responsible for enzymatic changes as the introduction of a charged aspartate residue at this location destabilizes the LID region, a flexible loop structure important in ATP binding and catalysis. This destabilization is thought to provide space for more bulky 3′-substituents, like those found in AZT, while negatively influencing feedback inhibitor interactions, such as those with dTTP [86]. The V84A mutation found in mutant B5 defines its altered substrate specificity as this amino acid exchange mimics the alanine found at the same position in human deoxycytidine kinase, and may be responsible for the increased preference towards deoxycytidine and its analogs [87].…”