Yeast peroxisomal multifunctional enzyme: (3R)-hydroxyacyl-CoA dehydrogenase domains A and B are required for optimal growth on oleic acid

The yeast peroxisomal (3R)-hydroxyacyl-CoA dehydrogenase/2-enoyl-CoA hydrat ase 2 (multifunctional enzyme type 2; MFE-2) has two N-terminal domains bel onging to the short chain alcohol dehydrogenase/reductase superfamily, To i nvestigate the physiological roles of these domains, here called A and B, S accharomyces cerevisiae fox-2 cells (devoid of Sc MFE-8) were taken as a mo del system. Gly(16) and Gly(329) Of the S. cerevisiae A and B domains, corr esponding to Gly16, which is mutated in the human MFE-2 deficiency, were mu tated to serine and cloned into the yeast expression plasmid pYE352, In ole ic acid medium, fox 2 cells transformed with pYE352:: ScMFE-2(a Delta) and pYE352::ScMFE-2(b Delta) grew slower than cells transformed with pYE352::Sc MFE-2, whereas cells transformed with pYE352::ScMFE-2(a Delta b Delta) fail ed to grow. Candida tropicalis MFE-2 with a deleted hydratase 2 domain (Ct MFE- 2(h2 Delta)) and mutational variants of the A and B domains (Ct MFE- 2 (h2 Delta a Delta), Ct MFE- 2(h2 Delta b Delta), and Ct MFE- 2(h2 Delta a D elta b Delta)) were overexpressed and characterized. All proteins were dime rs with similar secondary structure elements. Both wild type domains were e nzymatically active, with the B domain showing the highest activity with sh ort chain and the A domain with medium and long chain (3R)-hydroxyacyl-CoA substrates, The data show that the dehydrogenase domains of yeast MFE-2 hav e different substrate specificities required to allow the yeast to propagat e optimally on fatty acids as the carbon source.