Although previous studies have reported lower transglycosylation conversions when pyrimidine nucleobases were used as acceptors, a structural basis for this has not been explored. We surmised that the generally lower conversions to non-natural pyrimidine nucleoside products is due to the reduced residence time of the smaller pyrimidine nucleobase relative to a larger purine analogue in the active site of LlNDT-2, which in turn could lead to competing hydrolysis. Indeed, a recent study of the reaction kinetics of a related NDT derived from Chroococcidiopsis thermalis (CtNDT) revealed preference for purine nucleobase donors, which was largely driven by the KM. To shed further light on the substrate scope of LlNDT-2, we sought to obtain structures of the enzyme in complex with 2ʹ-deoxyribonucleosides. The structure of WT-LlNDT-2 (EC 2.4.2.6)