YerE-like enzymes for the synthesis of tertiary alcohols.

Carboligation reactions are highly valuable transformations in modern applied biocatalysis. Tertiary α-hydroxy ketones have broad relevance, yet their synthesis remains challenging. This 1,2-functionality can be introduced by thiamine diphosphate (ThDP)-dependent enzymes. However, most ThDP-dependent enzymes form new C-C bonds using aldehydes as acceptor substrates, yielding secondary α-hydroxy ketones. Only a few enzymes accept ketones as substrates, enabling access to chiral tertiary alcohols. Here, we describe a bioinformatic strategy to identify ThDP-dependent enzymes producing tertiary alcohols and present protocols for their expression, purification, and characterization. These protocols provide a framework for discovering ThDP-dependent enzymes that produce tertiary α-hydroxy ketones.