A single site mutation can induce functional promiscuity in homoserine kinase.

L-Homoserine kinase is crucial in the biosynthesis of L-threonine, L-isoleucine, and L-methionine, where it catalyzes ATP-dependent phosphorylation of L-homoserine (Hse) to yield L-homoserine phosphate as its native activity. However, a single site mutation of H138 → L shows the emergence of ATPase activity as a secondary function. However, a previous mechanistic study proposes direct involvement of ATP and the substrate without any catalytic base; therefore, how the mutation of H138 → L causes the secondary function remains an enigma. Using computational tools herein, we provide new insight into the catalytic mechanism of L-homoserine kinase, showing direct involvement of H138 as a catalytic base. We show that mutation of H138 → L opens a new water channel connecting ATP, which facilitates the ATPase activity and reduces the native activity. The proposed mechanism agrees with the experimental finding that an H138 → L mutation reduces the kinase activity but enhances a promiscuous function, ATPase activity. Since homoserine kinase catalyzes the biosynthesis of amino acids, we believe that an accurate mechanism could be significant for enzyme engineering to synthesize amino acid analogs.