Dictyostelium discoideum salvages purine deoxyribonucleosides by highly specific bacterial-like deoxyribonucleoside kinases.
The salvage of deoxyribonucleosides in the social amoeba Dictyostelium discoideum, which has an extremely A+T-rich genome, was investigated. All native deoxyribonucleosides were phosphorylated by D. discoideum cell extracts and we subcloned three deoxyribonucleoside kinase (dNK) encoding genes. D. discoideum thymidine kinase was similar to the human thymidine kinase 1 and was specific for thymidine with a K(m) of 5.1 microM. The other two cloned kinases were phylogenetically closer to bacterial deoxyribonucleoside kinases than to the eukaryotic enzymes. D. discoideum deoxyadenosine kinase (DddAK) had a K(m) for deoxyadenosine of 22.7 microM and a k(cat) of 3.7 s(-1) and could not efficiently phosphorylate any other native deoxyribonucleoside. D. discoideum deoxyguanosine kinase was also a purine-specific kinase and phosphorylated significantly only deoxyguanosine, with a K(m) of 1.4 microM and a k(cat) of 3 s(-1). The two purine-specific deoxyribonucleoside kinases could represent ancient enzymes present in the common ancestor of bacteria and eukaryotes but remaining only in a few eukaryote lineages. The narrow substrate specificity of the D. discoideum dNKs reflects the biased genome composition and we attempted to explain the strict preference of DddAK for deoxyadenosine by modeling the active center with different substrates. Apart from its native substrate, deoxyadenosine, DddAK efficiently phosphorylated fludarabine. Hence, DddAK could be used in the enzymatic production of fludarabine monophosphate, a drug used in the treatment of chronic lymphocytic leukemia.
Sandrini
Michael Paolo Bastner
MP
BioCentrum-DTU, Technical University of Denmark, Lyngby, Denmark. msandrini@aki.ku.dk <msandrini@aki.ku.dk>
Söderbom
Fredrik
F
Mikkelsen
Nils Egil
NE
Piskur
Jure
J
D000818:Animals; D000970:Antineoplastic Agents / Q000494:pharmacology; D002454:Cell Differentiation; D003853:Deoxyribonucleosides / Q000737:chemistry*; D004023:Dictyostelium / Q000235:genetics / Q000378:metabolism*; D005786:Gene Expression Regulation; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010766:Phosphorylation; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000378:metabolism*; D011687:Purines / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D014740:Vidarabine / Q000031:analogs & derivatives / Q000737:chemistry
D000970:Antineoplastic Agents; D003853:Deoxyribonucleosides; D011687:Purines; D011994:Recombinant Proteins; D017853:Phosphotransferases (Alcohol Group Acceptor); C020018:deoxyribonucleoside kinases; D014740:Vidarabine; C024352:fludarabine