Next, we performed preliminary in silico docking studies to rationalize why 1a is only converted by TtPyNP and not by other closely related and highly promiscuous enzymes such as GtPyNP. We hypothesized that conversion of this substrate would primarily be limited by steric hindrance during substrate binding, since i) uridine and 1a only differ by a single methyl group distant from the anomeric position and ii) TtPyNP displays significantly lower affinity for 1a than for uridine. PyNPs generally exhibit marked flexibility during their catalytic cycle with a transition from an open conformation to a closed state requiring a domain movement of approximately 8 Å. Since all first sphere residues in the closed state are highly conserved and identical between the tested PyNPs, we anticipated that initial binding in the open conformation would be a limiting factor, as TtPyNP offers slightly more space than GtPyNP due to a threonine-serine substitution at the back of the active site, as evident from sequence alignments. To examine this hypothesis, we obtained an X-ray crystal structure of GtPyNP at 1.9 Å resolution (see Supporting Information for details; PDB ID 7m7k) and used AutoDock Vina implemented in YASARA to dock uridine and 1a into the open conformations of this structure and the known Xray crystal structure of TtPyNP (PDB ID 2dsj). Docking of uridine and 1a into TtPyNP yielded structures in good agreement with the native mode of substrate binding via Hbonding to the nucleobase and positioning of the anomeric carbon near the phosphate binding pocket (Figures and). Likewise, uridine could be docked into GtPyNP in a similar position to the cocrystallized substrate (Figures and), where the 4ʹ-position of uridine is located in proximity to Thr84 (Ser83 in TtPyNP). However, we were unable to obtain sensible docking results for 1a with GtPyNP as the increased steric bulk at the 4ʹ-position consistently led to a rotation of the sugar scaffold into an unproductive pose (Figure ). This suggested that the subtle space-creating mutation to a serine in TtPyNP might be a key factor for conversion of 1a. Consistent with this conclusion, the slightly more sterically congested TtPyNP-S83T mutant significantly lost activity compared to the parent enzyme (kobs = 0.25 s -1 vs kobs = 0.59 s -1 , Table ), while the reverse substitution in GtPyNP installed a low but measurable level of activity in this enzyme (kobs = 0.02 s -1 for GtPyNP-T84S). Moreover, all other enzymes we screened initially, and which were inactive with 1a, also possess a threonine at this position, which likely impedes their ability to bind this substrate productively. Although such subtle but crucial space-creating mutations are rare, there is precedent from other enzymes in the literature. Together, these results indicate that sufficient space in the open conformation of PyNPs is a prerequisite for conversion of sterically more demanding substrates such as 1a. Clearly, there are other factors influencing the rate constant of this Figure . Phosphorolysis of 4ʹ-methyluridine (1a). The data for uridine in D were taken from ref. 26. Please see the Supporting Information for details and the externally hosted supplementary information for raw data. transformation, as evident from the order of magnitude difference between the rate constants of the active enzymes, but these must arise from mutations far from the active site, as all other residues in possible contact with the substrate are identical between the tested enzymes.