The lysine biosynthesis pathway occurs in bacteria, plants andsome fungi, but is absent in animals. In plants, lysine is one of themost limiting amino acids, raising considerable interest in developingstrategies to increase lysine content of agriculturally-important plants,such as wheat, maize and grapevine.In plants lysine production is controlled by the enzyme, dihydrodipicolinate synthase (DHDPS), which is regulated by lysine-mediatedallosteric feedback inhibition. However, the precise mechanism of allostery remains elusive. Recent studies suggest that a π-stacking interaction between Tyr131–Tyr132 represents the major communicationnetwork between the allosteric and active sites. In addition, Trp78is shown to flip towards bound ligand in the allosteric cleft, suggesting this residue acts as a “hydrophobic gate”, shielding bound lysinefrom solvent. This study tested these hypotheses by introducing pointmutations at positions 78 & 131.Trp78Ala, Tyr131Ala and Tyr131Phe mutants were expressed andpurified to homogeneity. Activity, lysine inhibition, and lysine binding propensity was assayed and compared to the wild type enzyme.Trp78Ala showed greatly attenuated lysine binding, consistent withits purported role in stabilising bound ligand. Tyr131Ala shows lowspecific activity, and attenuated lysine inhibition & binding affinity,suggesting this mutation abolishes the π-stack. Whilst Tyr131Phedisplays similar specific activity to the WT enzyme, suggesting it retains the π-stack, this mutant possesses attenuated lysine inhibitionand binding affinity, consistent with the loss of the hydroxyl, whichplays a role in stabilising bound allosteric ligand. These results support the project hypotheses, providing scope for the rational design ofgenetically-modified, high     lysine crops.