Shifting the NAD/NADP preference in class 3 aldehyde dehydrogenase

Among pyridine-nucleotide-dependent oxidoreductases, the class 3 family of aldehyde dehydrogenases (ALDHs) is unusual in its ability to function with either NAD or NADP. This is all the more surprising because an acidic resid ue, Glu140, coordinates the adenine ribose 2' hydroxyl. In many NAD-depende nt dehydrogenases a similarly placed carboxylate is thought to be responsib le for exclusion of NADP. The corresponding residue in most (approximate to 71%) sequences in the ALDH extended family is also Glu, and most of these are NAD-specific enzymes. Site-directed mutagenesis was performed on this r esidue in rat class 3 ALDH. Our results indicate that this residue contribu tes to tighter binding of NAD in the native enzyme, but suggest that additi onal factors must contribute to the ability to utilize NADP. Mutagenesis of an adjacent basic residue (Lys137) indicates that it is even more essentia l for binding both coenzymes, consistent with its conservation in nearly al l ALDHs (> 98%).