Enzyme’s Changing Shape Steers Molecular Recognition, Simulations Show

Enzymes are routinely described as molecular machines, but the phrase can be misleading. A machine implies a fixed chassis with moving parts, whereas many enzymes behave more like soft, breathing structures whose entire geometry shifts as they work. That flexibility is not a nuisance to be averaged away; it can be the very thing that decides which molecules the enzyme grabs, how long it holds them, and what chemistry it ultimately performs. A new computational study from Shibaura Institute of Technology in Japan has now put this idea on a quantitative footing for an industrially and medically important enzyme, showing that the protein’s shape alone can determine whether a bound molecule stays put or drifts away, and that a long-standing puzzle about sugar preferences dissolves once the enzyme is viewed in motion rather than frozen in a single snapshot.

The enzyme in question is pyrimidine-nucleoside phosphorylase, or PyNP, from the bacterium Bacillus subtilis. PyNP participates in the production of nucleic-acid building blocks by cleaving and reassembling nucleosides, the sugar-and-base units that form the letters of DNA and RNA. Because the same reaction chemistry can be redirected to process synthetic nucleoside analogues, the enzyme is a workhorse in the design of anticancer, antiviral, and antibacterial drugs, and it is also used to manufacture artificial nucleosides, fluorescently labeled nucleosides, and stable-isotope-labeled nucleic acids for research and diagnostics. Despite decades of practical use, the molecular basis of how PyNP’s changing shape governs molecular recognition has remained unclear, leaving a gap between what chemists observe at the bench and what is actually happening at atomic resolution inside the active site.

Addressing that gap, a research team led by Professor Akihiko Hatano, together with researcher Kousei Takeshima of the Course of Chemistry and Biotechnology, Department of Materials Science and Chemistry, College of Engineering at Shibaura Institute of Technology, set out to test how different conformations of the enzyme influence ligand retention and sugar preference. Their strategy was to treat the enzyme not as one structure but as a family of structures. Using all-atom molecular dynamics simulations, the team sampled four distinct conformational states of PyNP, probed the active site with 13 structural probes, and accumulated molecular dynamics trajectories totaling 15.6 microseconds of simulation time. The findings were published online in the journal ACS Omega on September 12, 2026.

The motivation, Professor Hatano explains, grew out of years of bench work. His group has long used this enzyme to synthesize artificial nucleosides, and a recurring question kept surfacing: why does changing the sugar component of a substrate alter the reaction? Experiments could document the outcomes, but they could not directly expose the atomic-scale motions responsible. “We wanted to understand this question from the perspective of atomic-scale motions that experiments alone cannot reveal,” said Prof. Hatano. “This led us to examine how the enzyme and its bound molecules behave across different structural states.” In other words, the team wanted to watch the enzyme and its cargo in real time at the level of individual atoms, rather than infer behavior from static crystal structures.

The first result concerns the geometry of the active-site pocket itself. As the protein transitions between its most closed and most open conformational states, the pocket that cradles the ligand expands by approximately 1.4-fold. That is a substantial change for a recognition site whose job is to grip molecules precisely. A pocket that breathes to this degree cannot be characterized by a single volume or a single set of contact distances; the microenvironment experienced by a bound nucleoside depends on which conformational state the enzyme happens to occupy at any moment. This observation alone suggests that docking studies and structure-based drug design built on one static structure may systematically misjudge how ligands behave in the real, dynamic enzyme.

The simulations then connected this breathing motion directly to function. Across the trajectories, 75.6 percent of those in the closed group retained their ligands in a bound state, compared with only 55.1 percent in the open group. The difference is striking: simply shifting the enzyme’s conformation changed the odds that a bound molecule would stay in place by roughly twenty percentage points. Ligand retention, in this view, is not a fixed property of the enzyme-ligand pair but a joint product of molecular identity and enzyme shape. A compound that appears loosely bound in an open structure might be firmly anchored in a closed one, and vice versa, which has immediate implications for how inhibitors targeting PyNP should be evaluated.

The study also resolved the sugar-preference puzzle. Ribose- and 2′-deoxyribose-containing compounds, it turns out, do not show a universal preference for one sugar over the other. Instead, sugar preference depended on both the individual compound and the enzyme’s conformation, meaning there is no single rule of thumb that predicts which sugar PyNP will handle better. The simulations did, however, reveal a consistent geometric signature: ribose occupied the North, or C3′-endo, ring conformation for 64.3 percent of the sampled time, whereas 2′-deoxyribose occupied the South, or C2′-endo, conformation for 57.9 percent. This conformational divergence was strongest for the unsubstituted compound and became progressively smaller as the substituent at the 6-position of the base grew larger, indicating that bulky base modifications can partially override the intrinsic tendencies of the two sugars.

A single amino acid residue emerged as a key player in this dynamic picture. Tyrosine 165, or Tyr165, behaves as a moving lid over the active site, an effect that was particularly pronounced in the closed reference structure. To test whether this lid genuinely contributes to ligand capture, the researchers ran additional simulations in which the residue was removed. Ligand retention dropped in those simulations, supporting the proposed lid mechanism at the computational level. The authors are careful to note a caveat: the strongest Tyr165 signal came mainly from a closed structure belonging to a related species, so confirmation using a closed-state structure from B. subtilis itself is still needed before the lid mechanism can be considered fully established for this organism.

The broader implications reach well beyond one bacterial enzyme. Because PyNP is already used to synthesize fluorescently labeled nucleosides and stable-isotope-labeled nucleic acids, understanding how enzyme shape affects molecular capture could help researchers design more efficient enzymatic synthesis routes and select reaction conditions that favor the desired conformational behavior. The same understanding could guide the design of inhibitors that exploit the closed state or the lid residue, a strategy relevant to anticancer and antiviral drug development. There is also a sustainability angle: enzymatic synthesis performed in water at mild temperatures is an inherently greener manufacturing route than many conventional chemical syntheses, and improving its efficiency through conformation-aware enzyme engineering could amplify that advantage.

“Enzymes should not be viewed as single, static structures when we think about molecular recognition,” said Prof. Hatano. “Our results show that different conformations can change how compounds are retained and how sugar-related preferences emerge.” The study points toward future calculations and experiments aimed at connecting these dynamic binding behaviors to the actual chemical steps of the phosphorylase reaction, a link that remains to be established. What the work already demonstrates, however, is methodological as much as mechanistic: examining multiple enzyme conformations with molecular simulations can reveal binding behavior that any single static structure may miss. By showing that ligand retention and sugar preference depend jointly on molecular identity and enzyme shape, the research provides a framework for dynamic substrate recognition and opens concrete paths toward future enzyme engineering, rational inhibitor design, and sustainable biocatalytic synthesis of the nucleoside analogues on which modern medicine and biological research increasingly depend.

Subject of Research: Conformation-dependent ligand retention and sugar recognition in pyrimidine-nucleoside phosphorylase from Bacillus subtilis studied by molecular dynamics simulations

Article Title: Molecular simulations reveal how an enzyme’s shape guides molecular recognition

Article References: Molecular simulations reveal how an enzyme’s shape guides molecular recognition. (n.d.). Original publication

Image Credits: AI Generated

DOI: Not provided

Keywords: pyrimidine-nucleoside phosphorylase, molecular dynamics simulations, Bacillus subtilis, molecular recognition, ligand retention, ribose, 2'-deoxyribose, active site, conformational states, enzyme engineering, nucleoside synthesis, ACS Omega