The crystal structure of the HSK with L-homoserine shows that H138 is situated at a large distance (7.3Å) from the catalytic site (i.e., from the reactive oxygen of the homoserine substrate (see Figure ). However, our study shows a significant conformational rearrangement in the active site residues during the MD simulation of the HSK with the substrate. In the simulated structure, the H138 residue, initially at 7.3Å from the catalytic site in the crystal structure, now comes closer to the catalytic site and maintains a very stable conformation of just 4Å from the catalytic site (see Figure ). At the same time, it forms a catalytic triad that includes H138, the polar OH group of the Lhomoserine, and the carboxylate group of D130. Interestingly, such triad formation was consistent over the entire simulation time of 100ns, and no further conformational changes were observed (see Figure ). We believe that the conformation of the active site seen in our simulation is the most catalytic relevant conformation. Due to crystal packing at cryogenic temperature, we might not have found the most catalytic relevant conformation in the X-ray crystallography, which might have led omission of the Histidine residue in the putative catalytic mechanism of the HSK enzymes. Based on the new conformation obtained during the MD simulations, we have proposed a new pathway of homoserine kinase-catalyzed phosphoryl transfer (see Scheme 1). As can be seen from Figure , H138 is well-positioned for proton abstraction from the hydroxyl group of L-homoserine, and therefore, it may abstract a proton from the hydroxyl group of Lhomoserine, triggering a nucleophilic attack upon the γ-phosphate of ATP. This results in the formation of a bond between γ-phosphate and the oxygen atom of L-homoserine. The resulting positive charge on H138 is further stabilized by nearby D140 residue. At the last stage of the mechanism, the abstracted proton is transferred from H138 to the phosphate, thus completing the reaction. Scheme 1: New proposed pathway to phosphoryl transfer in homoserine kinase.