DOMAINS OF THE TETRAFUNCTIONAL PROTEIN ACTING IN GLYOXYSOMAL FATTY-ACID BETA-OXIDATION - DEMONSTRATION OF EPIMERASE AND ISOMERASE ACTIVITIES ON A PEPTIDE LACKING HYDRATASE ACTIVITY

Peroxisomes from different eukaryotic organisms house a multifunctiona l protein acting in fatty acid beta-oxidation. In plant glyoxysomes, o ne of the isoforms of this protein contains the activities of L-3-hydr oxyacyl-CoA hydrolyase (EC 4.2.1.17), L-3-hydroxyacyl-CoA dehydrogenas e (EC 1.1.1.211), D-3-hydroxyacyl-CoA epimerase, and Delta(3),Delta(2) - enoyl-CoA isomerase (EC 5.3.3.8). This was demonstrated after molecu lar cloning of a cDNA coding for a protein of 79047 Da and its bacteri al expression. Chromatographic purification yielded a monomeric protei n exhibiting all four activities. In addition, mutant forms were prepa red, and peptides representing single domains were purified. Peptides containing the N-terminal region showed D-3-hydroxyacyl-CoA epimerase and Delta(3),Delta(2)-enoyl-CoA isomerase activities but lacked 2-tran s-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activities . Using the N-terminal fragment, we demonstrated that the D-3-hydroxya cyl-CoA converting activity is actually an epimerase rather than part of a combined water eliminating and water attaching system. The C-term inal half of the multifunctional protein represents the dehydrogenase domain.