Conformation- and Compound-Dependence of Ribose/2′-Deoxyribose Retention Patterns in Pyrimidine-Nucleoside Phosphorylase from Bacillus subtilis : A Four-Structure Molecular Dynamics Study

Abstract In pyrimidine-nucleoside phosphorylase (PyNP), how the conformational state shapes substrate recognition remains poorly understood. In this study, all-atom molecular dynamics (MD) with MM-PB(GB)SA decomposition was performed on four structures of PyNP from Bacillus subtilis spanning the conformational range: the open 5OLN, the semi-open 5EP8orig, an energy-minimized contracted (closed-like) model 5EP8 min, and the cross-species closed-state crystal 1BRW, the last added to define the closed-state end. Thirteen structural probes were used (ten purine-like ribose/2′-deoxyribose analogues and three inhibitors), sampling 156 trajectories and 15.6 μs in aggregate. All raw trajectories were deposited in a public repository (Zenodo, 10.5281/zenodo.21448920). The main results were threefold. (1) Sugar-ring puckering analysis showed that, overall, ribose preferred C3′-endo (north, 64%) and 2′-deoxyribose preferred C2′-endo (south, 58%) (p = 0.021). However, this per-sugar difference was largest for the unsubstituted sugar and shrank as the 6-position substituent became larger; sugar-ring puckering therefore provides a molecular correlate of sugar discrimination in this probe set, but its extent depends on the 6-position substituent. (2) Ligand retention was high at the closed-state end. However, the ribose-versus-2′-deoxyribose preference was not a property of the whole active site but a compound-by-compound and conformation-by-conformation property: averaged across compounds, and for many between-state differences, it was not statistically significant at the compound level. (3) Tyr165 behaved as a lid at the closed-state end; however, this signal arose mainly from the cross-species 1BRW structure and lost significance when 1BRW was excluded, so it requires confirmation with a closed-state structure of the same species. These retention metrics measure not equilibrium affinity but dynamic retention over 100 ns (the fraction of frames in which the ligand remained in the active site). This study clarifies how the retention, contact, and sugar preference of these structural probes depend on the conformational state and does not directly reproduce the experimentally reported ribose-donor selectivity. To test these conclusions, three sets of additional all-atom MD (each in three replicas) were performed. (i) The falsifiable mutant prediction was tested by Ala-mutant MD: Y165A, with the lid residue removed, showed significantly reduced ligand retention (BF_late median 0.02; one-sided p = 0.049 versus the control mutants retaining Tyr165). (ii) In a control MD explicitly including the co-substrate phosphate (HPO42–), the phosphate bound to the Lys cluster and could approach the anomeric carbon C1′ closely (contact fraction up to 96%; closest approach 2.81 Å), but the reactive in-line attack geometry required for substitution was essentially never adopted (below 0.5% of contact frames; 0% in 30 of the 33 systems), consistent with the interpretation that ribose-donor selectivity is decided at the transition state and is not accessible to equilibrium MD, pointing to the need for QM/MM or transition-state-level analysis. (iii) Extending the central systems to 300 ns gave retention and sugar-preference conclusions identical to the 100 ns analysis, supporting (rather than confirming) the conclusions obtained at the 100 ns scale; only a subset of central systems was extended, and only 46% of the original systems reached a plateau, so these extensions are reported as consistent with the 100 ns results. On this basis, three falsifiable mutagenesis predictions (Y165A, Q153A, K81A/K108A/K188A) are proposed.