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 threonineresidue (Thr175) immediately downstream of the
B strand in the Rossmann fold, the nucleotide specificityof Vh-ALDH has been changed from NADP
+ to NAD
+. Replacement of Thr175 by a negatively chargedresidue (Asp or Glu) resulted in an increase in
kcat/
Km for NAD
+ relative to that for NADP
+ of up to5000-fold due to a decrease for NAD
+ and an increase for NADP
+ in their respective Michaelis constants(
Ka). Differential protection by NAD
+ and NADP
+ against thermal inactivation and comparison of thedissociation constants of NMN, 2'-AMP, 2'5'-ADP, and 5'-AMP for these mutants and the wild-typeenzyme clearly support the change in nucleotide specificity. Moreover, replacement of Thr175 with polarresidues (N, S, or Q) demonstrated that a more efficient NAD
+-dependent enzyme T175Q could be createdwithout loss of NADP
+-dependent activity. Analysis of the three-dimensional structure of Vh-ALDHwith bound NADP
+ showed that the hydroxyl group of Thr175 forms a hydrogen bond to the 2'-phosphateof NADP
+. Replacement with glutamic acid or glutamine strengthened interactions with NAD
+ andindicated 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 residueat 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 withNADP
+ in Vh-ALDH.