Abstract
Three enzyme activities involved in fatty acid β-oxidation, i.e., those of enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and 3-oxoacyl-CoA thiolase, are exhibited by one multienzyme complex (HDT) composed of two molecules each of two peptides in Pseudomonas fragi. Using specific antisera against the two subunits of HDT, we isolated the genes encoding the subunits of HDT and designated them “faoA” (for the α-subunit) and “faoB” (for the β-subunit). Their complete nucleotide sequences were determined and it was revealed that faoA and faoB, both with individual putative S. D. sequences at suitable positions, formed a cluster, in that order. The amino acid sequences deduced from the nucleotide sequences of the two genes indicated that the α-subunit, encoded by faoA, is a polypeptide of 715 amino acid residues, and that the β-subunit, encoded by faoB, consists of 390 amino acid residues lacking the first methionine of the primary product encoded by faoB. Immunoblotting of cell lysates prepared from Escherichia coli transformants carrying plasmids which possess the faoA and/or faoB gene with antisera against the subunits of HDT showed that both the faoA and faoB genes were transcribed and translated in E. coli. The overall activities of 2-enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase were increased in the E. coli cells transformed with the plasmid possessing the faoA gene, suggesting that both the hydratase and dehydrogenase activities may be exhibited by the α-subunit of HDT. 3-Oxoacyl-CoA thiolase activity may be exhibited by the β-subunit as judged from the result of an amino acid homology search, but the α-subunit was necessary for an increase in the 3-oxoacyl-CoA thiolase activity of the β-subunit in E. coil.
