L-Threonine aldolases (TAs) represent a family of homologous pyridoxal 5’-phosphate-dependent enzymes found in bacteria and fungi, and catalyse the reversible cleavage of several l-3-hydroxy-α-amino acids. TAs have great biotechnological potential, since they catalyse the formation of carbon-carbon bonds, and therefore may be exploited for bioorganic synthesis of l-3-hydroxyamino acids that are biologically active or constitute building blocks for pharmaceutical molecules. Many TAs, showing different stereospecificity towards the Cβ configuration, have been isolated. Because of their potential to carry out diastereoselective syntheses, TAs have been the subject of structural, functional and mechanistic studies. Nevertheless, their catalytic mechanism and the structural bases of their stereospecificity have not been elucidated.
In this study, we have determined the crystal structure of low-specificity l-threonine aldolase from Escherichia coli at 2.2 Å resolution, in the unliganded form and co-crystallized with l-serine and l-threonine. Furthermore, several active-site mutants have been functionally characterised in order to elucidate the reaction mechanism and the molecular bases of stereospecificity. No active site catalytic residue was revealed, and a structural water molecule was assumed to act as catalytic base in the retro-aldol cleavage reaction.
Interestingly, the very large active site opening of E. coli TA suggests that a much larger molecule than l-threonine isomers may be easily accommodated, and threonine aldolases may actually play diverse physiological functions in different organisms. Substrate recognition and reaction specificity seem to be guided by the overall microenvironment that surrounds the substrate at the enzyme active site, rather than to one ore more specific residues.