Abstract
Understanding the structure-function relationships of pyridoxal-5'-phosphate (PLP)-dependent transaminases is key to advancing pyridoxal-phosphate-dependent catalysis and engineering transaminases for industrial applications. Despite our extensive knowledge of PLP-dependent enzymatic reactions, engineering transaminase activity and stability remains challenging. Here, we present the functional characterization of a novel PLP-dependent fold type IV transaminase from Desulfomonile tiedjei, alongside a detailed analysis of PLP binding and holoenzyme stability. This new transaminase exhibits activity toward various D-amino acids and (R)-phenylethylamine. Structural modeling and site-directed mutagenesis of residues in the second shell of the PLP-binding site revealed their roles in cofactor binding and the transaminase's catalytic efficiency. Notably, the T199Q variant demonstrated a fivefold increase in PLP affinity and improved activity under alkaline conditions. This is attributed to a newly formed hydrogen bond that stabilizes the N1-binding region of PLP. Glutamine at position 199 is not observed in homologous transaminases, making this non-natural substitution a novel and beneficial modification. These findings emphasize the importance of second-shell interactions in stabilizing PLP and expand our understanding of the structural diversity within PLP fold type IV transaminases. This paves the way for the engineering of more stable and versatile biocatalysts for industrial applications.