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Non-natural l -nucleoside analogues are increasingly used as therapeutic agents to treat cancer and viral infections. To be active, l -nucleosides need to be phosphorylated to their respective triphosphate metabolites. This stepwise phosphorylation relies on human enzymes capable of processing l -nucleoside enantiomers. We used crystallographic analysis to reveal the molecular basis for the low enantioselectivity and the broad specificity of human 3-phosphoglycerate kinase (hPGK), an enzyme responsible for the last step of phosphorylation of many nucleotide derivatives. Based on structures of hPGK in the absence of nucleotides, and bound to l and d forms of MgADP and MgCDP, we show that a non-specific hydrophobic clamp to the nucleotide base, as well as a water-filled cavity behind it, allows high flexibility in the …