Change of nucleotide specificity and enhancement of catalytic efficiency in single point mutants of Vibrio harveyi aldehyde dehydrogenase

The fatty aldehyde dehydrogenase from the luminescent bacterium, Vibrio har veyi (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 PB Strand in the Rossmann fold, the nucleotide specifici ty of Vh-ALDH has been changed from NADP(+) to NAD(+). Replacement of Thr17 5 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 Mich aelis constants (K-a). Differential protection by NAD(+) and NADP(+) agains t 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 cl early support the change in nucleotide specificity. Moreover, replacement o f Thr175 with polar residues (N, S, or Q) demonstrated that a more efficien t NAD(+)-dependent enzyme T175Q could be created without loss of NADP(+)-de pendent activity. Analysis of the three-dimensional structure of Vh-ALDH wi th bound NADP(+) showed that the hydroxyl group of Thr175 forms a hydrogen bond to the 2'-phosphate of NADP(+). Replacement with glutamic acid or glut amine strengthened interactions with NAD(+) and indicated why threonine wou ld be the preferred polar residue at the nucleotide recognition site in NAD P(+)-specific aldehyde dehydrogenases, These results have shown that the si ze and the structure of the residue at the nucleotide recognition site play the key roles in differentiating between NAD(+) and NADP(+) interactions w hile the presence of a negative charge is responsible for the decrease in i nteractions with NADP(+) in Vh-ALDH.