The fatty aldehyde dehydrogenase from the luminescent bacterium, Vibrio harveyi (Vh-ALDH), is unique with respect to its high specificity for NADP(+) over NAD(+). By mutation of a single threonine residue (Thr175) immediately downstream of the beta(B) strand in the Rossmann fold, the nucleotide specificity of Vh-ALDH has been changed from NADP(+) to NAD(+). Replacement of Thr175 by a negatively charged residue (Asp or Glu) resulted in an increase in k(cat)/K(m) for NAD(+) relative to that for NADP(+) of up to 5000-fold due to a decrease for NAD(+) and an increase for NADP(+) in their respective Michaelis constants (K(a)). Differential protection by NAD(+) and NADP(+) against thermal inactivation and comparison of the dissociation constants of NMN, 2'-AMP, 2'5'-ADP, and 5'-AMP for these mutants and the wild-type enzyme clearly support the change in nucleotide specificity. Moreover, replacement of Thr175 with polar residues (N, S, or Q) demonstrated that a more efficient NAD(+)-dependent enzyme T175Q could be created without loss of NADP(+)-dependent activity. Analysis of the three-dimensional structure of Vh-ALDH with bound NADP(+) showed that the hydroxyl group of Thr175 forms a hydrogen bond to the 2'-phosphate of NADP(+). Replacement with glutamic acid or glutamine strengthened interactions with NAD(+) and indicated why threonine would be the preferred polar residue at the nucleotide recognition site in NADP(+)-specific aldehyde dehydrogenases. These results have shown that the size and the structure of the residue at the nucleotide recognition site play the key roles in differentiating between NAD(+) and NADP(+) interactions while the presence of a negative charge is responsible for the decrease in interactions with NADP(+) in Vh-ALDH.