Though enzymes are very specific for their native reactions, they possess secondary functions to some extent . Such promiscuous functions are believed to be the root cause of the evolution of modern enzymes and their divergence from ancestral enzymes . Therefore, a mechanistic pathway of enzyme promiscuity has significant importance in the study of enzyme evolutions . Usually, the enzymes show promiscuity in the substrate selectivity, but they retain the nature of native reaction . Mechanistic studies of enzyme promiscuity show that some strategic mutations change the substrate recognition pattern, which, in turn, causes promiscuity in enzymes 8-10 . However, some special cases do also exist where a single site mutation causes functional diversity where an enzyme catalyzes an entirely new function without compromising its binding site topology 11- . Homoserine kinase (HSK) is an enzyme with such peculiarity. Natively, it catalyzes ATP-dependent phosphorylation of L-homoserine (Hse) to yield L-homoserine phosphate during the biosynthesis of L-threonine, L-isoleucine, and L-methionine 14, . We refer to it as 'kinase activity' (see Figure ). However, experiments, including the kinetic study, show that a single site mutation of Histidine to Leucine (H139L) in HSK of E.coli can efficiently catalyze 'ATPase activity' (150-fold higher than native activity), i.e., a water-mediated conversion of ATP to ADP and inorganic phosphate as a secondary function (see Figure ) . Interestingly, the mechanistic studies of the kinase activity of HSK propose no involvement of a Histidine or a base in the catalysis; instead, it is believed phosphoryl transfer reaction is initiated by the direct attack of the hydroxyl group of homoserine to γ-phosphate of ATP in the absence of base 14, . (see Figure ) However, it raises a mechanistic enigma: if HSK doesn't need a Histidine or a base for its catalytic activity, then how does the mutation of H139 result in the diversification of the catalytic function of HSK? Additionally, the H139 is conserved among all known HSK families; therefore, nature must have chosen it for some specific function. The present study aims to answer such intriguing questions by providing a new mechanistic route that shows a direct involvement of Histidine as a base. Using comprehensive MD simulations complemented by hybrid QM/MM (quantum mechanical molecular mechanical) calculation here, we show that H139 in E.coli (or H138 in M. jannaschii) has an evolutionary significance in the function of the HSK enzyme and, therefore, mutation of the H138L causes functional promiscuity in the enzyme.