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.