Multicyclic D-Stereospecific Hydrolase Dimer With High Sustained Activity.

Area of Science:

• Biocatalysis
• Protein Engineering
• Structural Biology

Background:

• Enzymes are crucial catalysts but often lack stability under industrial conditions like high temperatures or organic cosolvents.
• Sequence-based methods improve enzyme stability, but chemical protein engineering offers alternative strategies by modifying the natural amino acid repertoire.
• Tailoring enzyme function and robustness is essential for expanding their applications in various processes.

Purpose of the Study:

• To improve the stability and robustness of a D-stereospecific hydrolase using chemical protein engineering.
• To investigate the efficacy of in situ protein cyclization (INCYPRO) for enzyme stabilization.
• To explore the potential of macrocyclization for creating enhanced biocatalysts.

Main Methods:

• Application of the in situ cyclization of proteins (INCYPRO) technique to a D-stereospecific hydrolase.
• Site-specific macrocyclization to enhance enzyme resilience to thermal and cosolvent stress.
• X-ray crystallography to confirm the structure of cross-linked protein dimers.

Main Results:

• Site-specific macrocyclization significantly improved the enzyme's resilience to heat and cosolvent stress.
• An unexpected cross-linked protein dimer exhibited enhanced activity and thermal stability.
• Engineered multicyclic enzyme dimers with multiple cross-linking sites retained high activity and outperformed wild-type enzymes under stress.

Conclusions:

• Protein macrocyclization is a versatile strategy for stabilizing both monomeric and multimeric enzymes.
• INCYPRO provides a powerful route to engineer robust biocatalysts with improved functional properties.
• Chemical protein engineering, through macrocyclization, offers complementary approaches to enhance enzyme performance.