DescriptionPyridoxal-5’-phosphate (PLP) dependent enzymes are involved in the amino acids metabolism in all living organisms and catalyze a number of diverse chemicals reactions, such as decarboxylation, transamination, racemization, beta-elimination, carbon-carbon bond cleavage and formation. In spite of their catalytic versatility the PLP-dependent enzymes share common reaction mechanism and only few structural fold types. Using a comparative bioinformatic analysis of genetic and structural data of homologous proteins belonging to the aminotransferase family of PLP-dependent enzymes, we have investigated how the reaction diversity was created by Nature. We have analysed a set of proteins with different reaction specificities and identified the subfamily specific positions and structural elements responsible for the functional diversity of these enzymes. These positions were found to play an important role in the catalysis by creation a specific microenvironment around the cofactor and thus influencing the intrinsic reactivity of PLP. The selected specific positions were used as hotspots for the rational engineering of L-threonine aldolase to reveal the putative amino acid positions that mediate catalytic activity and substrate specificity in this class of enzymes. The obtained knowledge can be prerequisite for the engineering of PLP-dependent enzymes to create an efficient biocatalyst for the asymmetric synthesis of non-canonical amino acids. Thus, engineered L-threonine aldolase was applied for the stereoselective synthesis of various alpha-tetrasubstituted alpha-amino acids with e.e.L > 99% starting from aldehydes and the amino acid donors (Fig. 1). Moreover, performing site-directed mutagenesis on specific positions responsible for the reaction specificity, we could evolve the promiscuous activities of L-threonine aldolases, e.g. transamination and racemization.
|Period||11 Jul 2017|
|Event title||Biotrans2017: 13th International Symposium on Biocatalysis and Biotransformations|
|Degree of Recognition||International|