Document Detail


Reconstitution, morphology and crystallization of a fatty acid beta-oxidation multienzyme complex from Pseudomonas fragi.
MedLine Citation:
PMID:  9396725     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
The fatty acid beta-oxidation multienzyme complex from Pseudomonas fragi, HDT, exhibits predominantly the three enzymic activities of 2-enoyl-CoA hydratase (EC 4.2.1.17), 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) and 3-oxoacyl-CoA thiolase (EC 2.3.1.16). The HDT complex is encoded by the faoAB operon, consisting of the faoA and faoB genes that encode two individual constituents, the alpha-subunit and the beta-subunit. We have constructed Escherichia coli overexpression systems for the faoAB gene product (coexpression of the alpha- and beta-subunits), the alpha-subunit alone and the beta-subunit alone, and have purified the three respective products. Gel-filtration analysis revealed that the faoAB gene product forms a heterotetrameric structure, alpha2beta2, identical with the native HDT oligomeric state from P. fragi, whereas the alpha-subunit and beta-subunit individually form dimers. Electron microscopy demonstrated that each protein morphologically adopts the above oligomeric structures. The HDT complex, reconstituted in vitro from the isolated alpha- and beta-subunits, exhibits the three original enzymic activities and yields the same crystal as those from the native enzyme. CD measurements indicated that the alpha- and beta-dimers hardly alter their global conformations upon the formation of the HDT complex. Interestingly, the beta-dimer alone does not exhibit 3-oxoacyl-CoA thiolase activity, whereas the alpha-dimer alone exhibits both the 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase activities. These results suggest that the contact between the alpha- and beta-subunits is essential for the thiolase activity. We have identified several structurally important proteolytic sites within each subunit, which are protected in the intact heterotetrameric molecule. These findings allow the possible location of the interface between the two subunits, which should be crucial for the exhibition of thiolase activity.
Authors:
M Ishikawa; Y Mikami; J Usukura; H Iwasaki; H Shinagawa; K Morikawa
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Publication Detail:
Type:  Journal Article    
Journal Detail:
Title:  The Biochemical journal     Volume:  328 ( Pt 3)     ISSN:  0264-6021     ISO Abbreviation:  Biochem. J.     Publication Date:  1997 Dec 
Date Detail:
Created Date:  1998-02-02     Completed Date:  1998-02-02     Revised Date:  2012-11-16    
Medline Journal Info:
Nlm Unique ID:  2984726R     Medline TA:  Biochem J     Country:  ENGLAND    
Other Details:
Languages:  eng     Pagination:  815-20     Citation Subset:  IM    
Affiliation:
Biomolecular Engineering Research Institute, 6-2-3 Furuedai, Suita, Osaka 565, Japan.
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MeSH Terms
Descriptor/Qualifier:
3-Hydroxyacyl CoA Dehydrogenases / chemistry,  genetics,  metabolism
Acetyl-CoA C-Acyltransferase / chemistry,  genetics,  metabolism
Circular Dichroism
Crystallization
Dimerization
Electrophoresis, Polyacrylamide Gel
Enoyl-CoA Hydratase / chemistry,  genetics,  metabolism
Escherichia coli / genetics
Gene Expression
Microscopy, Electron
Multienzyme Complexes / chemistry*,  genetics,  metabolism*,  ultrastructure
Protein Conformation
Protein Structure, Secondary
Pseudomonas / enzymology*
Recombinant Proteins / chemistry,  isolation & purification,  metabolism
Trypsin / metabolism
Chemical
Reg. No./Substance:
0/Multienzyme Complexes; 0/Recombinant Proteins; 0/mitochondrial fatty acid beta-oxidation trifunctional protein; EC 1.1.1.35/3-Hydroxyacyl CoA Dehydrogenases; EC 2.3.1.16/Acetyl-CoA C-Acyltransferase; EC 3.4.21.4/Trypsin; EC 4.2.1.17/Enoyl-CoA Hydratase
Comments/Corrections

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