Profiling of Diverse Pyridoxal-5’-Phosphate Dependent Enzymes Reveals Promiscuous Aldolase Activity with (2-Azaaryl)methanamines

The elaboration of amine substrates through C–C bond forming reactions is important in the synthesis of bioactive small mole-cules. Pyridoxal-5'-phosphate (PLP)-dependent enzymes have emerged as valuable biocatalysts for this class of reactions, due to their high stereoselectivity and ability to forge new C–C bonds on unprotected α-amino acid substrates. However, the use of abiological primary amines as pronucleophiles with enzymes such as threonine aldolase has been unexplored, moderating the utility of a biocatalytic approach in the synthesis of diverse 1,2-amino alcohols. In this report, we disclose the discovery and engineering of a PLP-dependent aldolase which accepts (2-azaaryl)methanamines in an aldol-type transformation. The 1,2-amino alcohol products generated, which contain representative heteroaromatic pharmacophores, are delivered with control over both the diastereoselectivity and enantioselectivity in the C–C bond forming event. Protein engineering provided variants with improved binding affinity for the abiological substrate and decreased affinity for the native α-amino acid, overcoming inhibition of the abiotic reaction by components of lysate, a major challenge in reaction discovery with PLP-dependent enzymes such as threonine aldolases. This work represents the first known example of C–C bond formation on non-amino acid sub-strates with threonine aldolase and provides a platform for further development of complexity building biocatalytic reactions with abiotic amine substrates.