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

Technical Summary: Characterization of the Lysine-Forming Saccharopine Dehydrogenase LYS1 from Filamentous Fungi for Plant Food Fermentation

Problem Statement
Lysine is an essential amino acid, and its biosynthesis in fungi occurs via the conserved $\alpha$-aminoadipate (AAA) pathway. The final step of this pathway is catalyzed by LYS1 (EC 1.5.1.7), a reversible NAD$^+$/NADH-dependent oxidoreductase converting saccharopine to L-lysine and $\alpha$-ketoglutarate. While the LYS1 enzyme from the model yeast Saccharomyces cerevisiae has been characterized, there is a significant knowledge gap regarding homologs from filamentous fungi (e.g., Aspergillus, Penicillium, Rhizopus) which are critical for solid-state food fermentation processes like tempeh, miso, and cheese production. Existing literature suggests the reaction directionality is pH-dependent, yet kinetic characterizations of the forward (lysine-forming) reaction have largely been conducted under suboptimal neutral pH conditions. Furthermore, the biochemical and mechanistic differences between yeast and filamentous fungal LYS1 enzymes remain unexplored, limiting the ability to optimize lysine biosynthesis in plant-based fermentation strategies.

Methodology
The study employed a multi-faceted approach combining phylogenetics, recombinant protein expression, biophysical characterization, kinetic analysis, and molecular dynamics (MD) simulations:

• Sequence and Structural Analysis: LYS1 homologs were retrieved from 14 filamentous fungi genomes and compared to S. cerevisiae using BLASTp, multiple sequence alignment (MAFFT), and phylogenetic reconstruction (IQ-TREE). Three-dimensional structures were predicted using AlphaFold3, and structural similarity was quantified using the Local Distance Difference Test (LDDT) via Foldseek.
• Recombinant Expression: Genes for LYS1 from S. cerevisiae, Aspergillus nidulans, Penicillium roqueforti, and Rhizopus microsporus were codon-optimized and expressed. S. cerevisiae LYS1 was expressed in E. coli, while filamentous fungal homologs were expressed in Pichia pastoris. All proteins were purified via Ni-affinity chromatography.
• Biophysical and Kinetic Characterization:
  • pH Profile: Enzymatic activity was measured across pH 7–9 for both forward (saccharopine $\to$ lysine) and reverse (lysine $\to$ saccharopine) reactions.
  • Thermal Stability: Optimal reaction temperatures and melting temperatures ($T_m$) were determined using standard assays and nano differential scanning fluorimetry (nanoDSF).
  • Kinetics: Apparent kinetic parameters ($k_{cat}$, $K_M$, catalytic efficiency) for the forward reaction were derived from initial rate measurements at varying substrate (saccharopine) and cofactor (NAD$^+$) concentrations, fitted to the Michaelis-Menten equation. Activation energies ($E_a$) were calculated via Arrhenius plots.
• Molecular Dynamics (MD): Simulations (100 ns) were performed on apo, NAD$^+$-bound, saccharopine-bound, and ternary complex states. Key metrics included Root Mean Square Deviation (RMSD), Root Mean Square Fluctuation (RMSF) of the active site, and the frequency of frames where the distance between the NAD$^+$ C4 and saccharopine C8 atoms fell below catalytic thresholds (4.5, 4.0, and 3.6 Å). Principal Component Analysis (PCA) was applied to protein-ligand contact occupancies.

Key Results

• Sequence and Structural Conservation: Phylogenetic analysis revealed distinct clades separating S. cerevisiae from filamentous fungi, with Aspergillus and Penicillium grouping closely. Despite significant sequence divergence (approx. 50–55% similarity between filamentous fungi and yeast), the overall protein fold was highly conserved (LDDT > 0.9). Critical catalytic residues (Lys77, His96, Glu122) and structural elements were preserved across all homologs.
• pH and Thermodynamic Dependence: All homologs exhibited strong pH dependence. The forward reaction (lysine formation) was favored at alkaline pH (optimum at pH 9), while the reverse reaction peaked at neutral pH. Consistent with thermodynamic constraints, the reverse reaction displayed significantly higher $k_{cat}$ values than the forward reaction across all enzymes and pH conditions tested.
• Kinetic Differences:
  • S. cerevisiae LYS1 demonstrated the highest catalytic turnover ($k_{cat} \approx 2.34$ s$^{-1}$) and the lowest activation energy ($E_a \approx 13.1$ kJ/mol).
  • A. nidulans and P. roqueforti showed intermediate activity.
  • R. microsporus LYS1 exhibited significantly reduced activity ($k_{cat} \approx 0.05$ s$^{-1}$) and the highest activation energy ($E_a \approx 53.3$ kJ/mol). Despite lower $k_{cat}$, R. microsporus displayed lower apparent $K_M$ values for both substrates, suggesting tight binding but a catalytic bottleneck.
• Thermal Properties: S. cerevisiae LYS1 possessed the highest melting temperature ($T_m \approx 60^\circ$C) and optimal reaction temperature ($45^\circ$C). Filamentous fungal enzymes showed lower thermal stability and optima (35–40°C), correlating with the known growth temperatures of their source organisms.
• Molecular Dynamics Insights:
  • Conformational Sampling: More active homologs (S. cerevisiae, A. nidulans) sampled near-reactive geometries (NAD$^+$ C4 to saccharopine C8 distances < 4.0 Å) more frequently than the less active R. microsporus.
  • Flexibility: S. cerevisiae showed a more rigid backbone in the apo state, while filamentous fungi exhibited higher flexibility. Upon ternary complex formation, filamentous fungi showed a sharper decrease in active site RMSF (increased rigidification) compared to the yeast enzyme.
  • Contact Organization: PCA of residue-ligand contacts revealed distinct interaction patterns. R. microsporus showed the lowest number of persistent contacts with NAD$^+$, potentially hindering effective cofactor positioning. A specific substitution (Ala289Cys) in R. microsporus was identified as a potential factor disrupting contacts near the hydride transfer region.

Significance and Claims
The authors claim that this study provides a mechanistic and kinetic basis for understanding lysine biosynthesis in filamentous fungi relevant to food fermentation. Key claims include:

1. Biochemical Diversity: Despite high structural conservation, LYS1 enzymes from filamentous fungi are biochemically diverse, with R. microsporus exhibiting a distinct catalytic bottleneck compared to Ascomycete homologs.
2. Thermodynamic Constraints: The strong preference for the reverse reaction across all homologs suggests that the forward flux of lysine biosynthesis is thermodynamically unfavorable and likely relies on intracellular metabolite concentrations and pathway coupling to proceed.
3. Mechanistic Origin of Kinetic Differences: The observed kinetic divergence is attributed not to a loss of the catalytic scaffold, but to differences in the organization and stability of the ternary complex. Specifically, the frequency of productive protein-ligand contacts and the ability to sample catalytic distances are lower in the less active R. microsporus enzyme.
4. Implications for Fermentation: The data suggests that R. microsporus may have inherent limitations in lysine production capacity compared to Aspergillus or Penicillium species. Consequently, these findings may inform the selection of fungal strains for solid-state fermentations aimed at enhancing lysine content in plant-based foods. The study confirms that the forward reaction is favored in alkaline conditions, which may imply specific subcellular localization requirements (e.g., peroxisomes) for optimal pathway function in vivo.