Mutagenesis of Key Residues in the Binding Center of l‐ Aspartate‐β‐Semialdehyde Dehydrogenase from Escherichia coli Enhances Utilization of the Cofactor NAD(H)

l ‐Aspartate‐β‐semialdehyde dehydrogenase (ASADH) is a key enzyme in the aspartate pathway. In bacteria, ASADH is highly specific for the cofactor NADP + rather than NAD + . Limited information on cofactor utilization is available, and neither the wild‐type protein nor the available mutants could utilize NAD + efficiently. In this study, we identified several residues crucial for cofactor utilization by Escherichia coli ASADH ( ec ASADH) by mutating residues within the cofactor binding center. Among the investigated mutants, ec ASADH‐Q350N and ec ASADH‐Q350N/H171A, which exhibited markedly improved NAD + utilization, were further investigated by various biochemical approaches and molecular modeling. Relative to the wild type, the two mutants showed approximately 44‐fold and 66‐fold increases, respectively, in the constant k cat / K m of NAD + . As desired, they could also utilize NADH efficiently to synthesize l ‐homoserine in cascade reactions in vitro.