Beta-oxidation system of the filamentous fungus Neurospora crassa. Structural characterization of the trifunctional protein.
Treatment of the trifunctional protein from Neurospora crassa with various proteases produced almost identical patterns of proteolytic fragments. To study the structural features of the protein in more detail limited proteolysis with trypsin was carried out. Polyclonal antibodies were raised against three different tryptic fragments. With the help of immunological methods and amino-terminal sequence analysis we were able to monitor the sequential cleavage steps during proteolysis. Two major fragments (an amino-terminal one of 51 kDa and a carboxyl-terminal one of 46 kDa) were identified at the first cleavage step, dividing the 93-kDa subunit of the trifunctional protein almost in half. Additional proteolysis products, deriving from either half, were formed in subsequent proteolytic steps. Combining these results with those obtained from enzyme analysis of the proteolyzed protein, a domain structure of the trifunctional protein is proposed. According to our model each subunit of the tetrameric protein consists of at least two large domains, the amino-terminal one possessing 2-enoyl-CoA hydratase and L-3-hydroxyacyl-CoA dehydrogenase activity and the carboxyl-terminal one bearing 3-hydroxyacyl-CoA epimerase activity.
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 / Q000737:chemistry; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases; D004746:Enoyl-CoA Hydratase / Q000737:chemistry; D015151:Immunoblotting; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000302:isolation & purification; D009492:Neurospora crassa / Q000201:enzymology*; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000302:isolation & purification; D019998:Racemases and Epimerases / Q000737:chemistry
D009097:Multienzyme Complexes; D010446:Peptide Fragments; D015094:3-Hydroxyacyl CoA Dehydrogenases; D010450:Endopeptidases; D004746:Enoyl-CoA Hydratase; D019998:Racemases and Epimerases; C029601:3-hydroxybutyryl-CoA epimerase