Unlocking Asymmetric Michael Additions in an Archetypical Class I Aldolase by Directed Evolution

Area of Science:

• Biocatalysis and Enzyme Engineering
• Organic Chemistry
• Structural Biology

Background:

• Class I aldolases are biocatalysts for asymmetric aldol additions, producing chiral β-hydroxy-carbonyl compounds.
• Expanding aldolase utility to other C-C bond-forming reactions, like Michael additions, is a significant challenge.
• Directed evolution offers a powerful strategy for redesigning enzyme function and expanding catalytic scope.

Purpose of the Study:

• To redesign 2-deoxy-d-ribose-5-phosphate aldolase (DERA) for enantioselective Michael addition reactions.
• To enhance the catalytic activity and efficiency of DERA for non-native carboligation reactions.
• To elucidate the structural basis for the evolved catalytic mechanism.

Main Methods:

• Utilized 11 rounds of directed evolution to engineer DERA from *Escherichia coli*.
• Characterized the redesigned enzyme (DERA-MA) for its activity in Michael additions of nitromethane to α,β-unsaturated aldehydes.
• Performed crystallographic analysis to determine the structural basis of the enhanced activity.

Main Results:

• The engineered DERA-MA enzyme exhibited a 190-fold increase in catalytic activity compared to wild-type DERA.
• DERA-MA efficiently catalyzed enantioselective Michael additions, yielding pharmaceutically relevant chiral synthons.
• Structural analysis revealed a shift from an enamine-based to an iminium-based catalytic mechanism in DERA-MA.

Conclusions:

• Directed evolution successfully expanded the catalytic repertoire of a class I aldolase to include asymmetric Michael additions.
• The engineered DERA-MA functions as a proficient "Michaelase" with potential for biocatalytic applications.
• This study highlights opportunities for exploring iminium catalysis using DERA-derived catalysts.