Multicyclic D-Stereospecific Hydrolase Dimer With High Sustained Activity.

Enzymes are powerful catalysts for selective transformations but often suffer from limited stability under operational conditions such as elevated temperature or the presence of organic cosolvents. While sequence-based strategies have been widely used to improve stability, chemical protein engineering enables modifications beyond the natural amino acid repertoire thereby offering complementary routes to tailor enzyme function and robustness. Here, we apply the in situ cyclization of proteins (INCYPRO) to a D-stereospecific hydrolase with low intrinsic thermal stability. Site-specific macrocyclization substantially improved resilience to heat and cosolvent stress. Unexpectedly, we discovered a cross-linked protein dimer with enhanced activity and thermal stability. The complex structure was confirmed by x-ray crystallography. Extending the INCYPRO approach, we engineered a multicyclic enzyme dimer with a total of four cross-linking sites, which not only retained high activity under benign conditions but also outperformed the wild-type under stress. Our findings establish protein macrocyclization as a versatile strategy to stabilize both monomeric and multimeric enzymes, providing a powerful route to robust biocatalysts.