Engineering transketolase for stereoselective α-hydroxyketone synthesis.

Transketolase, a thiamine diphosphate-dependent enzyme, is widely distributed in nature and plays a crucial role in cellular metabolism. Its ability to synthesize α-hydroxyketones in a stereoselective manner, key precursors for high-value compounds like vicinal diols and amino alcohols, has garnered significant interest in synthetic chemistry. In this chapter, we review the engineering and applications of transketolase along with molecular docking studies, mutant library screening, and detailed experimental protocols. Engineering efforts have primarily focused on broadening substrate specificity for both donor and acceptor molecules, enhancing catalytic activity, improving stability, refining stereoselectivity, facilitating reverse cleavage reactions, and constructing novel covalent bonds. Advances in structural and computational analyses have deepened the understanding of the transketolase catalytic mechanism, guiding its engineering and significantly enhancing its industrial applicability. Current challenges in synthetic applications are also discussed to inform further optimization.