In contrast to the numerous interactions between the cytosine base and the aromatic slot, the ﬁve membered rings of 3TC and TRO make only limited enzyme contacts (Figures 3A and 4). Critical for catalysis is correct position- ing of the 50-OH, which receives the transferred phosphate group from ATP. In the L-nucleoside analog structures depicted in Figure 3A, the 50-OH group is hydrogen bonded to the side chain of Arg128. Consequently in each case, the O50-hydroxyl is close to a side chain oxygen atom of Glu53 (3.1–3.6 s), the general base that activates 50-OH group attack of the g -phosphate of ATP (see Figures 2B and 3). To achieve this critical interaction, concomitant with base tilting, the ﬁve-membered rings of 3TC and TRO rotate 30 around the glycosidic bond (c, marked with an arrow in Figure 2B) relative to the dCK-bound conformation of D-dC. A comparable c rotation observed in the L-dC structure (E. Sabini, S. Hazra, M. Konrad and A. Lavie, unpublished data) provides additional evidence that our structural ﬁndings represent the way in which L-nucleosides and L-nucleoside analogs bind to dCK. The four structures depicted in Figure 3 document that positioning of the 50-OH group in the active site of dCK is independent of chirality. This remarkable property derives from the internal symmetry and conformational ﬂexibility (i.e. rotation about the glycosidic torsion angle c) of nucleo- sides and nucleoside analogs. Once the cytosine base is docked into the aromatic slot, the paucity of protein–ligand interactions involving the ﬁve-membered ring and the confor- mational properties of the nucleoside permit appropriate posi- tioning of the 50-OH group. Within the aromatic slot, possible glycosidic bond torsion angles are limited by the hydrophobic residues Ile30 and Leu82 (Figures 2C and 4). An additional degree of freedom dictating the precise location of the hydroxyl is the pucker of the ﬁve-membered ring. For 3TC and TRO, the ring pucker is equivalent to C30 endo, whereas for L-dC and D-dC the sugar pucker is C30 exo. This differ- ence can be attributed in part to the absence of a 30-OH group on 3TC and on TRO, both of which can act as DNA chain terminators. Finally, a torsion angle rotation about the C40 and C50 bond, from gauche in the D-dC structure to trans in the complex structures of 3TC, TRO and L-dC, direct the 50-OH group towards Glu53. Although these features of 3TC and TRO conformational ﬂexibility bring the 50-OH group near to Glu53 (3.1–3.6 s; Figure 3A), they do not stabilize the very close, so-called catalytic, interactions seen with L-dC and D-dC (2.4–2.5 s; Figure 3B). However, the fact that dCK shows similar catalytic efﬁciency towards 3TC/ TRO and L-dC (Table 2) suggests that the slightly sub- optimal position observed for the 50-OH group in the 3TC/ TRO structures does not impede catalysis. In conclusion, the structures reported herein document that dCK phosphorylation of L-nucleosides and L-nucleoside ana- logs is due to both the nature of the enzyme active site and the nature of the substrates. We suggest that lack of enan- tiomeric selectivity may reﬂect the central role dCK plays in nucleotide salvage, i.e. phosphorylating both the pyrimi- dine dC and the purines dA and dG within the cytosolic com- partment. The by-product of such promiscuity appears to be non-enantioselectivity. Similar arguments can be invoked to explain why the related enzymes dGK, which phosphorylates dA and dG, and TK2, which phosphorylates dT and dC, Nucleic Acids Research, 2007, Vol. 35, No. 1 191 are not enantioselective. In contrast, human TK1, which is restricted to D-dT phosphorylation, is exquisitely enantiose- lective (25). We suggest that enzymes that evolved to catalyze more than one chemically distinct nucleoside substrate acquired less restrictive active sites, which in turn endowed such enzymes with hitherto unexpected catalytic activities for substrates with non-physiologic chirality. ACKNOWLEDGEMENTS The authors thank the SERCAT staff for help in data collection, and Dr Chung K. (David) Chu for a troxacitabine sample and helpful discussions. The authors acknowledge Dr Gilles Gosselin for providing a sample of L-dC. This work was supported by the NIH (E.S., S.H. and A.L.) and the Max Planck Society (M.K.). 3TC was obtained through the AIDS Research and Reference Reagent Program, Division of AIDS, NIAID, NIH. Funding to pay the Open Access publication charges for this article was provided by NIH grant RO1 CA95687. Conflict of interest statement. One of the authors (S.K.B.) is an employee of SGX Pharmaceuticals, which is testing the compound troxacitabine as a therapeutic agent. REFERENCES 1. 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