A Double Residue Substitution in the Coenzyme-binding Site Accounts for the Different Kinetic Properties between Yeast and Human Formaldehyde Dehydrogenases

Abstract

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 M) 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 ).Formaldehyde is a highly reactive compound that is present in the environment as a result of natural processes and human industrial activity. Moreover, formaldehyde is also generated endogenously by several metabolic pathways (1-5). In humans the level of formaldehyde in blood has been estimated as 0.46 -2.81 M (6). The elimination of formaldehyde in eukaryotic cells is mainly carried out by glutathione-dependent formaldehyde dehydrogenase (FALDH) 1 (7). Formaldehyde reacts spontaneously with glutathione (GSH) to form the S-hydroxymethylglutathione (S-HMGSH) adduct which, in the presence of NAD ϩ , is oxidized to S-formylglutathione by FALDH (8). S-Formylglutathione is irreversibly hydrolyzed by S-formylglutathione hydrolase to formate and GSH (8). In addition to the glutathionedependent formaldehyde dehydrogenase activity, FALDH is also active with long chain alcohols, particularly with -hydroxylated fatty acids such as 12-hydroxydodecanoic acid (9 -12). Interestingly FALDH is capable of catalyzing efficiently the NADH-dependent degradation of S-nitrosoglutathione derived from the glutathione nitrosation by nitric oxide (13). FALDH is a dimeric enzyme with subunits of 40 kDa, containing two zinc atoms per subunit, and it appears to be ubiquitous in eukaryotic organisms (14). FALDH is a member of the medium chain alcohol dehydrogenase (ADH) family, and it represents the ADH class (class III) most closely related to the ancestral line that originated by successive g...

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