Characterization of aspartokinase double mutants using a combination of experiments and simulations.

Aspartokinase (AK) is synergistically suppressed by Thr and Lys in the Corynebacterium metabolic pathway. Site-directed mutations can significantly improve AK inhibition. Our previous studies confirmed that sites 379 and 380 were important sites affecting enzyme activity, so we further screen the double mutants with excellent enzymatic properties from sites 379 and 380, and discuss the difference of enzyme activity between the double mutants and single mutants. Here, a double mutant, T379L/A380 M, with improved enzyme activity (2.74-fold) was obtained. Enzymatic property experiments showed that the optimum temperature of T379L/A380 M increased from 26 °C (recombinant Escherichia coli; WT-AK) to 45 °C and that the optimal pH decreased from 8.0 (WT-AK) to 7.5. Further, the half-life decreased from 4.5 to 3.32 h. These enzymatic properties were better than other mutant strains. Inhibition was diminished with low concentrations of Lys, and Lys + Thr presented an activating role. Subsequently, the reasons for the improved AK enzyme activity were illustrated with microscale thermophoresis (MST) experiments and molecular dynamic (MD) simulation by measuring ligand affinity and AK conformational changes. MST showed that the affinity between T379L/A380 M and Lys decreased, but the affinity between T379L/A380 M and Asp increased, promoting T379L/A380 M enzyme activity. MD experiments showed that T379L/A380 M enhanced the Asp-ATP affinity and catalyzed the transfer of residues S192 and D193 to Asp, promoting T379L/A380 M enzyme activity. However, the mutation did not cause fluctuations in the substrate Asp and ATP pockets. This might be why the enzyme activity was inferior to that of the single mutants (T379L and A380 M).

Quinoa husk peptides reduce melanin content via Akt signaling and apoptosis pathways.

To improve the treatment of pigmentation disorders, looking for natural and safe inhibitors of melanin synthesis has become an area of research interest. The quinoa husk peptides reportedly elicit various biological activities (e.g., anti-cancer, antioxidant, anti-hypertensive, and so forth), but its effects on melanin inhibition remain unknown. In the current study, we purified quinoa husk peptides with 30 and 80% ethanol using a macroporous adsorption resin (DA201-C). Component screening revealed that the 80%-ethanol fraction (i.e., QHP fraction) contained numerous short peptides (84.41%) and hydrophobic amino acids (45.60%), while eliciting a superior tyrosinase [TYR]-inhibition rate, 2,2-diphenyl-1-picryhydrazil-scavenging rate, reducing activity, and chelating capacity compared to the 30% fraction and was thus applied in subsequent analyses. Differentially expressed genes in the QHP fraction were primarily enriched in the Akt-signaling pathways based on transcriptomics. Thus, we assessed the expression of related proteins and genes in A375 cells and rat skin cells following treatment with QHP.