Mimicking enzyme evolution by generating new (betaalpha)8-barrels from (betaalpha)4-half-barrels.

Area of Science:

• Biochemistry
• Structural Biology
• Enzyme Engineering

Background:

• Gene duplication and fusion are key mechanisms in enzyme evolution.
• The (betaalpha)8-barrel fold, common in enzymes, likely evolved from smaller (betaalpha)4-half-barrel domains.
• Understanding this evolutionary process can inform the design of new enzymes.

Purpose of the Study:

• To experimentally mimic the proposed evolutionary pathway of (betaalpha)8-barrel enzymes.
• To generate novel (betaalpha)8-barrel proteins in vitro using (betaalpha)4-half-barrel domains.
• To explore the potential for enzyme diversification through domain exchange.

Main Methods:

• Deduced (betaalpha)4-half-barrels from imidazole glycerol phosphate synthase (HisF) and isomerase (HisA).
• Generated tandem duplications (HisF-CC) and optimized protein structures (HisF-C*C).
• Created chimeric proteins (HisFA, HisAF) by crosswise gene fusion of half-barrels.

Main Results:

• HisF-CC showed increased stability compared to HisF-C.
• HisF-C*C formed a stable, monomeric (betaalpha)8-barrel.
• The chimeric protein HisAF yielded a stable, compact (betaalpha)8-barrel monomer with cooperative unfolding.
• HisFA exhibited native secondary structures but unstable associations.

Conclusions:

• The study successfully generated novel (betaalpha)8-barrel proteins in vitro by mimicking evolutionary duplication and fusion events.
• Results demonstrate that domain exchange of (betaalpha)4-half-barrels can lead to new (betaalpha)8-barrels with potentially novel enzymatic functions.
• This provides a new strategy for enzyme diversification and engineering.