Primary structures of the genes, faoA and faoB, from Pseudomonas fragi B-0771 which encode the two subunits of the HDT multienzyme complex involved in fatty acid beta-oxidation.
Three enzyme activities involved in fatty acid beta-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 alpha-subunit) and "faoB" (for the beta-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 alpha-subunit, encoded by faoA, is a polypeptide of 715 amino acid residues, and that the beta-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 alpha-subunit of HDT.(ABSTRACT TRUNCATED AT 250 WORDS)
Sato
S
S
Department of Biology, College of Arts and Sciences, University of Tokyo.
Hayashi
M
M
Imamura
S
S
Ozeki
Y
Y
Kawaguchi
A
A
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000235:genetics*; D000102:Acetyl-CoA C-Acyltransferase / Q000235:genetics*; D000595:Amino Acid Sequence; D001483:Base Sequence; D004746:Enoyl-CoA Hydratase / Q000235:genetics*; D005227:Fatty Acids / Q000378:metabolism*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D010084:Oxidation-Reduction; D011549:Pseudomonas / Q000201:enzymology / Q000235:genetics*; D016415:Sequence Alignment
D005227:Fatty Acids; D009097:Multienzyme Complexes; D015094:3-Hydroxyacyl CoA Dehydrogenases; D000102:Acetyl-CoA C-Acyltransferase; D004746:Enoyl-CoA Hydratase