Nucleic Acids Research Advance Access published online on July 10, 2007
Nucleic Acids Research, doi:10.1093/nar/gkm479
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Nucleic Acid Enzymes |
Enantioselectivity of human AMP, dTMP and UMP-CMP kinases
1Laboratoire d’Enzymologie Moléculaire, FRE 2852-CNRS-Université Paris 6, 4, place Jussieu, 75005 Paris 2Institut des Biomolécules Max Mousseron (IBMM), UMR 5247 CNRS-Universités Montpellier 1 et 2, case courrier 1705, Bâtiment Chimie 17, Université Montpellier 2, Place Eugène Bataillon, 34095 Montpellier cedex 5 and 3Unité de Chimie Organique, URA CNRS 2128, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris Cedex15, France
*To whom correspondence should be addressed. Tel: +33 1 44 27 59 93, Fax: +33 1 44 27 59 94; Email: ddeville{at}ccr.jussieu.fr
Received April 27, 2007. Revised May 30, 2007. Accepted May 31, 2007.
L-Nucleoside analogues such as lamivudine are active for treating viral infections. Like D-nucleosides, the biological activity of the L-enantiomers requires their stepwise phosphorylation by cellular or viral kinases to give the triphosphate. The enantioselectivity of NMP kinases has not been thoroughly studied, unlike that of deoxyribonucleoside kinases. We have therefore investigated the capacity of L-enantiomers of some natural (d)NMP to act as substrates for the recombinant forms of human uridylate-cytidylate kinase, thymidylate kinase and adenylate kinases 1 and 2. Both cytosolic and mitochondrial adenylate kinases were strictly enantioselective, as they phosphorylated only D-(d)AMP. L-dTMP was a substrate for thymidylate kinase, but with an efficiency 150-fold less than D-dTMP. Both L-dUMP and L-(d)CMP were phosphorylated by UMP-CMP kinase although much less efficiently than their natural counterparts. The stereopreference was conserved with the 2'-azido derivatives of dUMP and dUMP while, unexpectedly, the 2'-azido-D-dCMP was a 4-fold better substrate for UMP-CMP kinase than was CMP. Docking simulations showed that the small differences in the binding of D-(d)NMP to their respective kinases could account for the differences in interactions of the L-isomers with the enzymes. This in vitro information was then used to develop the in vivo activation pathway for L-dT.
The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors.
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