Fatty aldehyde dehydrogenase (ALDH) from the luminescent marine bacterium, Vibrio harveyi, differs from other ALDHs in its unique specificity and high affinity for NADP+. Two glutamic acid residues, Glu253 and Glu377, which are highly conserved in ALDHs, were investigated in the present study. Mutation of Glu253 to Ala decreased the kcat for ALDH activity by over four orders of magnitude without a significant change in the K(m) values for substrates or the ability to interact with nucleotides. Both thioesterase activity and a pre-steady-state burst of NAD(P)H were also eliminated, implicating Glu253 in promoting the nucleophilicity of the cysteine residue(Cys289) involved in forming the thiohemiacetal intermediate in the enzyme mechanism. Mutation of Glu377 to Gln (E377Q mutant) selectively decreased the kcat for NAD(+)-dependent ALDH activity (> 10(2)-fold) compared to only a 6-fold loss in NADP(+)-dependent activity without comparable changes to the K(m) values for substrates. Consequently, the E377Q mutant had a very high specificity for NADP+(kcat/K(m) > 10(3) of that for NAD+) which was over 20 times higher than that of the wild-type ALDH. Although a pre-steady-state burst of NAD(P)H was eliminated by this mutation, thioesterase activity was completely retained. Using [1-2H]acetaldehyde as a substrate, a significant deuterium isotope effect was observed, implicating Glu377 in the hydride transfer step and not in acylation or release of the acyl group from the cysteine nucleophile. The increase in specificity of the E377Q mutant for NADP+ is consistent with a change in the rate-limiting step determining kcat from nucleotide-dependent NAD(P)H dissociation to hydride transfer. The results provide biochemical evidence that the two highly conserved Glu residues are involved in different functions in the active site of V. harveyi ALDH.