Directed Evolution of a Protein Catenane Reveals How Noncanonical Topology Modulates Sequence–Property Relationships

Using single-domain catenated dihydrofolate reductase (<i>cat</i>-DHFR) as a model, we combined homologue expansion, directed evolution, and topology reversion to compare matched linear and catenated isoforms. Catenation proved broadly compatible with DHFR homologues, yet soluble yield and catalytic activity remained strongly sequence dependent.

Directed evolution substantially improved the catalytic activity of <i>cat</i>-DHFR through enhanced substrate binding and turnover, while largely preserving or increasing thermal stability. Strikingly, reverting evolved catenanes to their linear counterparts transferred these functional gains: all reverted linear isoforms exhibited higher catalytic activity and thermal stability than wild-type DHFR. Across matched topological pairs, catenation generally increased thermal stability, whereas catalytic activity depended more strongly on sequence background and topology.