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 catalyzes the oxidation of the fatty aldehyde substrate for the light-emitting reaction. On the basis of the amino-terminal sequence of Vh.ALDH, a degenerate probe was used to screen a genomic library of V harveyi in pBR322, a positive clone was selected containing the Vh.ALDH gene and expressed in Escherichia coli, and the enzyme was purified to homogeneity. Although the sequence of the V. harveyi ALDH significantly diverged from other aldehyde dehydrogenases, mutation of a conserved cysteine implicated in catalysis completely inactivated the enzyme without loss of its ability to bind nucleotides, consistent with a catalytic 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 (Km = 1.4 microM) over 40 times lower than that reported for other aldehyde dehydrogenases. Although V. harveyi aldehyde dehydrogenase is unique in terms of its high specificity for NADP+, the identification of a catalytic conserved cysteine in Vh.ALDH clearly indicates that a highly related mechanism and structure have been retained among even the most diverged aldehyde dehydrogenases.
Vedadi
M
M
Department of Biochemistry, McGill University, Montreal, Canada.
Szittner
R
R
Smillie
L
L
Meighen
E
E
D000445:Aldehyde Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003545:Cysteine / Q000737:chemistry; D004269:DNA, Bacterial / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D007700:Kinetics; D008163:Luminescent Measurements; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009249:NADP / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D014733:Vibrio / Q000201:enzymology* / Q000235:genetics
D004269:DNA, Bacterial; D011994:Recombinant Proteins; D009249:NADP; D000445:Aldehyde Oxidoreductases; C038828:long-chain-aldehyde dehydrogenase; D003545:Cysteine