A double residue substitution in the coenzyme-binding site accounts for the different kinetic properties between yeast and human formaldehyde dehydrogenases.
Glutathione-dependent formaldehyde dehydrogenase (FALDH) is the main enzymatic system for formaldehyde detoxification in all eukaryotic and many prokaryotic organisms. The enzyme of yeasts and some bacteria exhibits about 10-fold higher k(cat) and K(m) values than those of the enzyme from animals and plants. Typically Thr-269 and Glu-267 are found in the coenzyme-binding site of yeast FALDH, but Ile-269 and Asp-267 are present in the FALDH of animals. By site-directed mutagenesis we have prepared the T269I and the D267E mutants and the D267E/T269I double mutant of Saccharomyces cerevisiae FALDH with the aim of investigating the role of these residues in the kinetics. The T269I and the D267E mutants have identical kinetic properties as compared with the wild-type enzyme, although T269I is highly unstable. In contrast, the D267E/T269I double mutant is stable and shows low K(m) (2.5 microM) and low k(cat) (285 min(-1)) values with S-hydroxymethylglutathione, similar to those of the human enzyme. Therefore, the simultaneous exchange at both residues is the structural basis of the two distinct FALDH kinetic types. The local structural perturbations imposed by the substitutions are suggested by molecular modeling studies. Finally, we have studied the effect of FALDH deletion and overexpression on the growth of S. cerevisiae. It is concluded that the FALDH gene is not essential but enhances the resistance against formaldehyde (0.3-1 mM). Moreover, the wild-type enzyme (with high k(cat) and K(m)) provides more resistance than the double mutant (with low k(cat) and K(m)).
Fernández
M R
MR
Department of Biochemistry, Universitat Autònoma de Barcelona, E-08193 Bellaterra (Barcelona), Spain.
Biosca
J A
JA
Torres
D
D
Crosas
B
B
Parés
X
X
D000445:Aldehyde Oxidoreductases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D001483:Base Sequence; D001665:Binding Sites; D003067:Coenzymes / Q000378:metabolism*; D017931:DNA Primers; D004795:Enzyme Stability; D006801:Humans; D007700:Kinetics; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D003067:Coenzymes; D017931:DNA Primers; D000445:Aldehyde Oxidoreductases; C087006:formaldehyde dehydrogenase, glutathione-independent