Enzyme-catalyzed approaches using aldolases are important tools for the asymmetric carbon-carbon bond formation and synthesis of polyfunctional compounds. Threonine aldolases (TA) are pyridoxal 5’-phosphate (PLP) dependent enzymes, which catalyze the reversible aldol reaction of glycine as donor with an aldehyde acceptor to form b-hydroxy-a-amino acids –important building blocks of many bioactive compounds and pharmaceuticals. These enzymes are highly selective at the α-carbon of the amino acid product. Both, L- and D-specific threonine aldolase activities have been found and various microbial enzymes have been isolated and biochemically characterized so far.
Most of the threonine aldolases accept a wide range of aliphatic and aromatic aldehydes as acceptor substrates but only glycine as donor substrate. Recently, we have found natural threonine aldolases, which are able to accept alanine as donor, thus chiral a-quaternary a-amino acids can be synthesized in one step with excellent stereoselectivity at a-carbon. One of our main research areas involves the development of TA catalysed synthesis of a diverse range of non-natural tertiary amine acids starting from simple prochiral aldehyde precursors, in a highly sustainable and stereoselective manner. An important aspect of this involves the expanding of biocatalyst toolbox, engineering the enzymes to accept other amino acid donors and improvement of the stereoselectivity at b-carbon of products as well as reaction yield by combination with additional (chemo)enzymatic steps.
We also focus on the understanding the detailed mechanism of TA catalysis by comparison it with other PLP-dependent enzymes and analysing the function of active site residues.
Pyridoxal-5’-phosphate (PLP), the active form of Vitamin B6, is a versatile cofactor used by more than 160 different enzymes classified by the Enzyme Commission. PLP-dependent enzymes are mainly involved in the amino acids metabolism in all living organisms and catalyze a number of diverse chemicals reactions, such as decarboxylation, transamination, racemization, b-elimination, carbon-carbon bond cleavage and formation. Due to their catalytic versatility, the PLP-enzymes have been exploited as biocatalysts for production of natural and non-natural amino acids and their related compounds. The remarkable versatility of vitamin B6-dependent enzymes makes them ideal candidates for investigation of their structural and functional relationships in order to modify their catalytic properties and broaden the number of biocatalysts applicable for biotransformations in organic synthesis.
Enzymes from large families often exhibit diverse functions and/or broad substrate ranges. Using comparative bioinformatic analysis of genetic and structural data of homologous proteins, we endeavour to understand how the functional diversity was created by natural evolution in the family of pyridoxal-5’-phosphate (PLP) dependent enzymes. This knowledge will be prerequisite to design the efficient biocatalysts, which can be applied for the asymmetric biocatalytic synthesis of non-canonical amino acids.