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 multifunctional protein acting in fatty acid beta-oxidation. In plant glyoxysomes, one of the isoforms of this protein contains the activities of L-3-hydroxyacyl-CoA hydrolyase (EC 4.2.1.17), L-3-hydroxyacyl-CoA dehydrogenase (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 molecular cloning of a cDNA coding for a protein of 79047 Da and its bacterial expression. Chromatographic purification yielded a monomeric protein exhibiting all four activities. In addition, mutant forms were prepared, 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-trans-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activities. Using the N-terminal fragment, we demonstrated that the D-3-hydroxyacyl-CoA converting activity is actually an epimerase rather than part of a combined water eliminating and water attaching system. The C-terminal half of the multifunctional protein represents the dehydrogenase domain.
Preisig-Müller
R
R
Department of Biochemistry, Philipps-University, Marburg, Federal Republic of Germany.
Gühnemann-Schäfer
K
K
Kindl
H
H
D015094:3-Hydroxyacyl CoA Dehydrogenases; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D019748:Carbon-Carbon Double Bond Isomerases; D003001:Cloning, Molecular; D017931:DNA Primers; D064006:Dodecenoyl-CoA Isomerase; D004746:Enoyl-CoA Hydratase / Q000378:metabolism*; D004926:Escherichia coli / Q000235:genetics; D005227:Fatty Acids / Q000378:metabolism*; D007535:Isomerases / Q000378:metabolism*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D015388:Organelles / Q000378:metabolism*; D010084:Oxidation-Reduction; D063994:Peroxisomal Bifunctional Enzyme; D019998:Racemases and Epimerases / Q000378:metabolism*; D051381:Rats; D017386:Sequence Homology, Amino Acid; D014675:Vegetables / Q000378:metabolism
D017931:DNA Primers; D005227:Fatty Acids; D009097:Multienzyme Complexes; D015094:3-Hydroxyacyl CoA Dehydrogenases; D004746:Enoyl-CoA Hydratase; D063994:Peroxisomal Bifunctional Enzyme; D007535:Isomerases; D019998:Racemases and Epimerases; C029601:3-hydroxybutyryl-CoA epimerase; D019748:Carbon-Carbon Double Bond Isomerases; D064006:Dodecenoyl-CoA Isomerase