Improving nucleoside diphosphate kinase for antiviral nucleotide analogs activation.
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.
Gallois-Montbrun
Sarah
S
Régulation Enzymatique des Activités Cellulaires, CNRS FRE 2364, Institut Pasteur, 25, rue du Dr. Roux 75724, Paris cedex 15, France.
Schneider
Benoit
B
Chen
Yuxing
Y
Giacomoni-Fernandes
Veronique
V
Mulard
Laurence
L
Morera
Solange
S
Janin
Joel
J
Deville-Bonne
Dominique
D
Veron
Michel
M
D000212:Acyclovir / Q000494:pharmacology; D000818:Animals; D000998:Antiviral Agents / Q000494:pharmacology; D001216:Asparagine / Q000494:pharmacology; D001665:Binding Sites; D002455:Cell Division; D004023:Dictyostelium / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006801:Humans; D007700:Kinetics; D008956:Models, Chemical; D008958:Models, Molecular; D009154:Mutation; D009701:Nucleoside-Diphosphate Kinase / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D016133:Polymerase Chain Reaction; D011485:Protein Binding; D012694:Serine / Q000378:metabolism; D013696:Temperature; D014162:Transfection
D000998:Antiviral Agents; D012694:Serine; D006639:Histidine; D001216:Asparagine; D009701:Nucleoside-Diphosphate Kinase; D000212:Acyclovir