Characterization of the lysine-forming saccharopine dehydrogenase Lys1 from filamentous fungi

Lysine biosynthesis in fungi proceeds through the α-aminoadipate pathway with Lys1 (EC 1.5.1.7) catalyzing the final reversible L-lysine producing step. Although Lys1 from Saccharomyces cerevisiae has been characterized, limited information is available on this highly conserved enzyme in filamentous fungi relevant for solid-state food fermentation. Here, we compare Lys1 homologs from Aspergillus nidulans, Penicillium roqueforti, and Rhizopus microsporus with the S. cerevisiae enzyme as reference using phylogenetic, biophysical, kinetic, and molecular dynamics analyses. Sequence and structural comparisons showed sequence divergence across fungal lineages while overall fold and key catalytic residues were conserved. All enzymes displayed strong directional pH dependence, with the forward L-lysine producing reaction favored at alkaline pH and the reverse reaction promoted at neutral pH. Analyses of temperature profile revealed homolog-specific differences, with the homolog from S. cerevisiae presenting the highest temperature optimum and melting temperature. The turnover (kcat) of the reverse reaction was consistently higher than the forward reaction for all homologs, consistent with thermodynamic constraints on lysine formation. Kinetic characterization revealed that the S. cerevisiae enzyme had the highest kcat, followed by A. nidulans and P. roqueforti, whereas the R. microsporus enzyme had lower activity and highest activation energy. Molecular dynamics simulations corroborated that the more active homologs more frequently sampled near-reactive NAD⁺ C4-saccharopine C8 geometries, while R. microsporus showed lower sampling frequency of these conformations and weaker protein–ligand contact organization. These findings provide a mechanistic and kinetic basis for understanding lysine biosynthesis in filamentous fungi. Key points • Phylogenetic, biophysical, kinetic, molecular dynamics of Lys1 from fungi • Catalytic residues and Lys1 folds are conserved (LDDT > 0.9) but sequences differ • Reaction direction (saccharopine ↔ L-lysine) is strongly dependent on pH