Aldehyde dehydrogenases play crucial roles in the detoxification of exogenous andendogenous aldehydes by catalyzing their oxidation to carboxylic acid counterparts. This study reports characterization of two such isoenzymes from the yeast Saccharomyces cerevisiae var. boulardii (NCYC 3264), one mitochondrial (Ald4p) and one cytosolic (Ald6p). Both Ald4p and Ald6p were oligomeric in solution and demonstrated positive kinetic cooperativity towards aldehyde substrates. Wild-type Ald6p showed activity only with aliphatic aldehydes. Ald4p, on the contrary, showed activity with benzaldehyde along with a limited range of aliphatic aldehydes.Inspection of modelled structure of Ald6p revealed that a bulky amino acid residue (Met-177, compared to the equivalent residue Leu-196 in Ald4p) might cause steric hindrance of cyclic substrates. Therefore, we hypothesized that specificities of the two isoenzymes towards aldehyde substrates was partly driven by steric hindrance in the active site. A variant of wildtype Ald6p with the Met-177 residue replaced by a valine was also characterized to address to the hypothesis. It showed an increased specificity range and a gain of activity towards cyclohexanecarboxaldehyde. It also demonstrated an increased thermal stability when compared with both the wild- types. These data suggest that steric bulk in the active site of yeast aldehydedehydrogenases is partially responsible for controlling specificity.
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