Structural basis for substrate recognition in l-lysine 6-dehydrogenase from Geobacillus stearothermophilus by Cryo-EM

L-lysine 6-dehydrogenase (LysDH; EC 1.4.1.18) oxidatively deaminates the ε-amino group of L-lysine. Due to its high substrate specificity, LysDH serves as a valuable tool for L-lysine quantification. However, the molecular basis of this specificity has remained unclear because of the lack of substrate-bound structures. In this study, we determined the cryo-electron microscopy (cryo-EM) structures of LysDH from the thermophilic bacterium Geobacillus stearothermophilus (GstLysDH) in the apo form at 2.9 Å resolution and in complex with NAD⁺ and L-lysine at 2.5 Å resolution. GstLysDH assembles as a tetramer, which undergoes a global conformational transition upon NAD⁺ binding. Structural analysis revealed that the α-carboxyl and α-amino groups of L-lysine were coordinated by oppositely charged residues, thereby orienting the ε-amino group toward the nicotinamide ring of NAD⁺ and anchoring the substrate in the optimal binding mode. This precise recognition mechanism accounts for the enzyme's strict specificity for the ε-amino group of L-lysine. Furthermore, comparative structural analysis with L-phenylalanine dehydrogenase suggests that the oxidative deamination in GstLysDH proceeds through a conserved hydride transfer mechanism. Together, these insights establish a structural framework for the rational design and industrial application of LysDH and related amino acid dehydrogenases.