Association of both enoyl coenzyme A hydratase and 3-hydroxyacyl coenzyme A epimerase with an active site in the amino-terminal domain of the multifunctional fatty acid oxidation protein from Escherichia coli.
An Escherichia coli mutant multienzyme complex of fatty acid oxidation, composed of two 41-kDa beta-subunits and two 79-kDa mutant alpha-subunits with the alpha/Gly116-->Phe substitution, has been overproduced and purified. The catalytic properties of 3-ketoacyl-coenzyme A (CoA) thiolase and L-3-hydroxyacyl-CoA dehydrogenase were found to be virtually identical with those of the wild type, whereas both enoyl-CoA hydratase and 3-hydroxyacyl-CoA epimerase activities were eliminated by the alpha/Gly116-->Phe mutation. delta 3-cis-delta 2-trans-Enoyl-CoA isomerase was only slightly affected by the mutation. The results of this study, together with the sequence analysis of the large alpha-subunit of the E. coli complex (Yang, X.-Y. H., Schulz, H., Elzinga, M., and Yang, S.-Y. (1991) Biochemistry 30, 6788-6795) and a demonstration of the epimerization of D-3-hydroxyacyl-CoAs in E. coli via a dehydration/hydration mechanism (Smeland, T. E., Cuebas, D., and Schulz, H. (1991) J. Biol. Chem. 266, 23904-23908), lead to the conclusion that enoyl-CoA hydratase and 3-hydroxyacyl-CoA epimerase are associated with a common active site in the amino-terminal domain of the multifunctional fatty acid oxidation protein. Thus the E. coli hydratase and epimerase activities represent two functions of a unique crotonase that converts both L- and D-3-hydroxyacyl-CoAs to 2-trans-enoyl-CoAs. Moreover, the results suggest that the amino-terminal domain of the large alpha-subunit is also involved in the isomerase activity but the key residue(s) required for catalyzing the isomerization is distinct from the crotonase.
Yang
S Y
SY
Laboratory of Neurobiochemistry, New York State Institute for Basic Research in Developmental Disabilities, Staten Island 10314.
Elzinga
M
M
D000102:Acetyl-CoA C-Acyltransferase / Q000378:metabolism*; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D004269:DNA, Bacterial; D004591:Electrophoresis, Polyacrylamide Gel; D004746:Enoyl-CoA Hydratase / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology*; D005227:Fatty Acids / Q000378:metabolism; D007700:Kinetics; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000378:metabolism*; D010084:Oxidation-Reduction; D019998:Racemases and Epimerases / Q000378:metabolism*; D013552:Swine
D004269:DNA, Bacterial; D005227:Fatty Acids; D009097:Multienzyme Complexes; D000102:Acetyl-CoA C-Acyltransferase; D004746:Enoyl-CoA Hydratase; D019998:Racemases and Epimerases; C029601:3-hydroxybutyryl-CoA epimerase