Engineering stereoselectivity of ThDP-dependent enzymes

Area of Science:

• Biochemistry and enzymology
• Organic chemistry
• Synthetic biology

Background:

• Thiamine diphosphate-dependent enzymes are vital biocatalysts involved in diverse C-C bond transformations.
• Decarboxylase and transketolase families exhibit distinct stereoselectivity (R- vs. S-selective) in their reactions.
• Understanding and controlling enzyme stereoselectivity is key for synthesizing enantiomerically pure compounds.

Purpose of the Study:

• To review recent advancements in stereoselective C-C bond formation catalyzed by thiamine diphosphate-dependent enzymes.
• To explore the mechanistic basis of stereoselectivity in these enzymes, focusing on benzoin condensation-like reactions.
• To highlight the creation of enzyme variants with altered stereoselectivity for novel synthetic applications.

Main Methods:

• Structure-function analyses employing site-directed mutagenesis to modify enzyme active sites.
• Investigation of substrate scope and reaction mechanisms for engineered enzyme variants.
• Assessment of the impact of different substrates and organic co-solvents on reaction stereoselectivity.

Main Results:

• Development of S-selective variants of pyruvate decarboxylase, benzoylformate decarboxylase, and SEPHCHC synthase.
• Creation of R-selective transketolase variants.
• Demonstrated access to a wide array of enantiocomplementary α-hydroxyketones and α,α'-dihydroxyketones.

Conclusions:

• Engineered thiamine diphosphate-dependent enzymes provide powerful tools for stereoselective synthesis.
• Mechanistic insights derived from structure-function studies guide the rational design of biocatalysts.
• These enzymes offer sustainable and efficient routes to valuable chiral building blocks.