Deciphering the evolution of flavin-dependent monooxygenase stereoselectivity using ancestral sequence reconstruction

Area of Science:

• Biocatalysis and Enzyme Engineering
• Natural Product Synthesis
• Structural Biology

Background:

• Controlling reaction selectivity is crucial for targeted synthesis, but challenging in biocatalysis due to enzymes' inherent preferences.
• Achieving complementary selectivity profiles in biocatalytic reactions is essential for divergent synthetic strategies.

Purpose of the Study:

• To investigate the structural features governing stereoselectivity in oxidative dearomatization reactions vital for azaphilone natural product synthesis.
• To understand how enzymes achieve tunable selectivity for complex molecule synthesis.

Main Methods:

• Utilized crystal structures of enantiocomplementary biocatalysts to hypothesize selectivity-controlling features.
• Employed ancestral sequence reconstruction (ASR) and resurrection to probe residue impacts on stereochemical outcomes.
• Combined ASR with mutational and computational studies.

Main Results:

• Identified two distinct mechanisms controlling stereochemical outcomes: one involving multiple active site residues (AzaH), the other a single residue switch (TropB and AfoD).
• Flavin-dependent monooxygenases (FDMOs) employ flexible and simple strategies to control stereoselectivity.
• Demonstrated the production of stereocomplementary azaphilone natural products by fungi.

Conclusions:

• ASR and resurrection are powerful tools for understanding enzyme mechanisms and guiding protein engineering.
• Enzymes utilize adaptable strategies to control stereoselectivity, enabling the synthesis of complex natural products.
• This research provides a foundation for future protein engineering efforts aimed at tunable biocatalysis.