Preparation and characterization of a bifunctional aldolase/kinase enzyme: a more efficient biocatalyst for C-C bond formation.

A bifunctional aldolase/kinase enzyme named DLF has been constructed by gene fusion through overlap extension. This fusion enzyme consists of monomeric fructose-1,6-bisphosphate aldolase (FBPA) from Staphylococcus carnosus and the homodimeric dihydroxyacetone kinase (DHAK) from Citrobacter freundii CECT 4626 with an intervening linker of five amino acid residues. The fusion protein was expressed soluble and retained both kinase and aldolase activities.

Substrate channelling in an engineered bifunctional aldolase/kinase enzyme confers catalytic advantage for C-C bond formation.

A new bifunctional enzyme that displays both aldolase and kinase activities has been designed and successfully used in the synthesis of aldol adducts, employing DHA as initial donor, with an increase in the reaction rate of 20-fold over the parent enzymes, which can be interpreted in terms of substrate channelling.