The beta-oxidation system in catalase-free microbodies of the filamentous fungus Neurospora crassa. Purification of a multifunctional protein possessing 2-enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities.
A trifunctional beta-oxidation protein, designated TFP, was purified to apparent homogeneity from oleate-induced mycelia of Neurospora crassa. 2-Enoyl-CoA hydratase, L-3-hydroxyacyl-CoA dehydrogenase, and 3-hydroxyacyl-CoA epimerase activities copurified in constant ratios with this protein when crude extracts were subjected to cation-exchange, dye-ligand, and adsorption chromatography. Trifunctionality was substantiated by coinciding enzyme activity ratios during the last two purification steps and additional chromatographic steps. The enzyme was shown to be a 365-kDa tetramer of subunits with a molecular mass of 93 kDa. Several lines of evidence suggest that these subunits are identical. Monospecific antibodies raised against the homogenous protein specifically precipitated the three enzymatic activities of TFP. Immunoblotting of fractions obtained after sucrose density gradient centrifugation of a crude extract indicated that TFP was exclusively localized in glyoxysome-like microbodies. The beta-oxidation system of N. crassa is structurally related to those of peroxisomes despite the presence of an acyl-CoA dehydrogenase rather than an acyl-CoA oxidase. A mitochondrial 2-enoyl-CoA hydratase activity was separated from TFP and purified to apparent homogeneity. The absence of all other beta-oxidation activities from mitochondria suggests that this organelle and its 2-enoyl-CoA hydratase are not involved in fatty acid degradation in N. crassa.
Thieringer
R
R
Institut für Physiologische Chemie, Abteilung für Zellbiochemie, Ruhr-Universität Bochum, Federal Republic of Germany.
Kunau
W H
WH
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D002374:Catalase / Q000032:analysis; D002851:Chromatography, High Pressure Liquid; D015180:Electrophoresis, Gel, Two-Dimensional; D004746:Enoyl-CoA Hydratase / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D008830:Microbodies / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009492:Neurospora crassa / Q000201:enzymology*; D010084:Oxidation-Reduction; D019998:Racemases and Epimerases / Q000302:isolation & purification* / Q000378:metabolism; D051381:Rats
D009097:Multienzyme Complexes; D015094:3-Hydroxyacyl CoA Dehydrogenases; D002374:Catalase; D004746:Enoyl-CoA Hydratase; D019998:Racemases and Epimerases; C029601:3-hydroxybutyryl-CoA epimerase