Cloning, mapping, and expression of genes involved in the fatty acid-degradative multienzyme complex of Escherichia coli.
Two protein subunits (42,000 and 78,000 daltons) encoded by the fadAB genes form a multifunctional enzyme complex containing thiolase, 3-hydroxyacyl-coenzyme A dehydrogenase, crotonase , epimerase, and isomerase activities (S. Pawar and H. Schulz, J. Biol. Chem. 256:3894-3899, 1981). In an attempt to characterize the structural organization and regulatory properties of these genes, a 5.2-kilobase pair fragment containing the fadAB genes has been isolated. Plasmids containing this fragment (i) complement mutations in the fadAB genes; (ii) overproduce by 10- to 50-fold thiolase, 3-hydroxyacyl-coenzyme A dehydrogenase and crotonase ; and (iii) specify a 42,000- and a 78,000-dalton protein. The fadA gene, which encodes the 42,000-dalton protein, has been localized within the original clone to a 3.3-kilobase pair fragment. Thiolase activity, which is encoded by the 42,000-dalton protein, was not observed in the absence of the 78,000-dalton protein, suggesting that an intact complex is required for function. Transposon Tn5 insertional mutagenesis of the cloned fadAB genes has demonstrated that both fadA and fadB are transcribed as a single transcriptional unit with the direction of transcription from fadA to fadB . The molecular cloning and characterization of the fadAB region confirm the original genetic contention that the genes encoding the proteins for the multifunctional complex form an operon.
D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000235:genetics; D000101:Acetyl-CoA C-Acetyltransferase / Q000235:genetics; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004262:DNA Restriction Enzymes; D004251:DNA Transposable Elements; D004746:Enoyl-CoA Hydratase / Q000235:genetics; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics*; D005227:Fatty Acids / Q000378:metabolism*; D005798:Genes, Bacterial; D005816:Genetic Complementation Test; D009097:Multienzyme Complexes / Q000235:genetics*; D009876:Operon; D014158:Transcription, Genetic
D004251:DNA Transposable Elements; D005227:Fatty Acids; D009097:Multienzyme Complexes; D015094:3-Hydroxyacyl CoA Dehydrogenases; D000101:Acetyl-CoA C-Acetyltransferase; D004262:DNA Restriction Enzymes; D004746:Enoyl-CoA Hydratase