INVOLVEMENT OF CYSTEINE-289 IN THE CATALYTIC ACTIVITY OF AN NADP(-SPECIFIC FATTY ALDEHYDE DEHYDROGENASE FROM VIBRIO-HARVEYI())

Fatty aldehyde dehydrogenase (Vh.ALDH) from the luminescent bacterium, Vibrio harveyi, may be implicated in controlling luminescence as it c atalyzes the oxidation of the fatty aldehyde substrate for the light-e mitting reaction. On the basis of the amino-terminal sequence of Vh.AL DH, a degenerate probe was used to screen a genomic library of V harve yi in pBR322, a positive clone was selected containing the Vh.ALDH gen e and expressed in Escherichia coli, and the enzyme was purified to ho mogeneity. Although the sequence of the V. harveyi ALDH significantly diverged from other aldehyde dehydrogenases, mutation of a conserved c ysteine implicated in catalysis completely inactivated the enzyme with out loss of its ability to bind nucleotides, consistent with a catalyt ic role for this residue. Using absorption and fluorescence assays for NAD(P)H, it was shown that NAD(+) and NADP(+) bound to the same site and that saturation of Vh.ALDH with NADP(+) occurred with a Michaelis constant (K-m = 1.4 mu M) over 40 times lower than that reported for o ther aldehyde dehydrogenases. Although V. harveyi aldehyde dehydrogena se is unique in terms of its high specificity for NADP(+), the identif ication of a catalytic conserved cysteine in Vh.ALDH clearly indicates that a highly related mechanism and structure have been retained amon g even the most diverged aldehyde dehydrogenases.