Abstract
Antiviral nucleoside analog therapies rely on their incorporation by viral DNA polymerases/reverse transcriptase leading to chain termination. The analogs (3'-deoxy-3'-azidothymidine (AZT), 2',3'-didehydro-2',3'-dideoxythymidine (d4T), and other dideoxynucleosides) are sequentially converted into triphosphate by cellular kinases of the nucleoside salvage pathway and are often poor substrates of these enzymes. Nucleoside diphosphate (NDP) kinase phosphorylates the diphosphate derivatives of the analogs with an efficiency some 10(4) lower than for its natural substrates. Kinetic and structural studies of Dictyostelium and human NDP kinases show that the sugar 3'-OH, absent from all antiviral analogs, is required for catalysis. To improve the catalytic efficiency of NDP kinase on the analogs, we engineered several mutants with a protein OH group replacing the sugar 3'-OH. The substitution of Asn-115 in Ser and Leu-55 in His results in an NDP kinase mutant with an enhanced ability to phosphorylate antiviral derivatives. Transfection of the mutant enzyme in Escherichia coli results in an increased sensitivity to AZT. An x-ray structure at 2.15-A resolution of the Dictyostelium enzyme bearing the serine substitution in complex with the R(p)-alpha-borano-triphosphate derivative of AZT shows that the enhanced activity reflects an improved geometry of binding and a favorable interaction of the 3'-azido group with the engineered serine.
Highlights
Nucleotide analogs such as dideoxynucleosides, AZT,1 and d4T are widely used in clinics for their antiviral effects, in
Whereas the first two phosphorylation steps are catalyzed by enzymes specific for the nucleobase, the ␥-phosphate is added by nucleoside diphosphate (NDP) kinase, which exhibits little specificity toward the nucleobase and the sugar moiety (1)
The substitution of a serine at the Asn-119 (Dictyostelium) or Asn-115 position improves the capacity of NDP kinase to use as substrates nucleotide analogs lacking a 3Ј-OH
Summary
Chemicals—Natural nucleotides (NDP and NTP) and dideoxynucleoside triphosphates were from Roche Molecular Biochemicals. Expression and Purification of Wild-type and Mutated NDP Kinases—Human NDPK-A mutants were obtained by PCR method using the overlap extension strategy. The wild type and mutant human NDPK-A were expressed and purified according to Ref. 10. Wild type and mutant Dd-NDPK were obtained as described previously (6) except for the N119Y mutant, which was partially purified by Q-Sepharose FF chromatography. Steady-state Kinetic Experiments—The activity of NDP kinase was measured at 20 °C with ATP and dTDP as substrates using coupled enzymes (pyruvate kinase and lactate dehydrogenase) (12). For enzymes with a ping-pong mechanism, the ratio of apparent kcat/Km measured at a given concentration of the other substrate is equal to the true value of kcat/Km. Stopped-flow Kinetic Experiments—As the diphosphate form was not always available, we used the triphosphate forms of the analogs to study phosphate transfer in half-reaction (Reaction 1).
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