Characterization of the lysine forming saccharopine dehydrogenase LYS1 from filamentous fungi for plant food fermentation

<title>Abstract</title> <p> 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 <italic>Saccharomyces cerevisiae</italic> 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 <italic>Aspergillus nidulans</italic> , <italic>Penicillium roqueforti</italic> , and <italic>Rhizopus microsporus</italic> with the <italic>S. cerevisiae</italic> 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 <italic>S. cerevisiae</italic> presenting the highest temperature optimum and melting temperature. The turnover ( <italic>k</italic> _cat ) 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 <italic>S. cerevisiae</italic> enzyme had the highest <italic>k</italic> _cat , followed by <italic>A. nidulans</italic> and <italic>P. roqueforti</italic> , whereas the <italic>R. microsporus</italic> 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 <italic>R. microsporus</italic> 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. </p>