Transketolase mutants previously identified for use with the non-phosphorylated aldehyde propanal have been explored with a series of linear and cyclic aliphatic aldehydes, and excellent stereoselectivities observed.

The uses of 3-formylbenzoic acid and 4-formylbenzoic acid as molecular probes along with previous and new transketolase mutants revealed the factors governing the rate of reaction between transketolase and aromatic aldehydes. The novel α,α-dihydroxyketones were produced at 15 to 30-fold higher yields and up to 250-fold higher specific activities with D469T TK when compared to those obtained for benzaldehyde.

Highlights► Previous single mutants of transketolase improved activity on new substrates. ► Recombination to form double mutants led to critical loss of functional expression. ► Mutated sites were found to be in a structural network of co-evolved residues. ► The network was re-adapted around previous mutants for kinetic synergy and functional expression.