Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase

Paul N Black, Concetta C DiRusso, Amy K. Metzger, Tamra L. Heimert

Research output: Contribution to journalArticle

134 Citations (Scopus)

Abstract

In the enteric bacterium, Escherichia coli, acyl coenzyme A synthetase (fatty acid:CoA ligase (AMP-forming) EC 6.2.1.3) activates exogenous long-chain fatty acids concomitant with their transport across the inner membrane into metabolically active CoA thioesters. These compounds serve as substrates for acyl-CoA dehydrogenase in the first step in the process of β-oxidation. The acyl-CoA synthetase structural gene, fadD, has been identified on clone 6D1 of the Kohara E. coli gene library and by a process of subcloning and complementation analyses shown to be contained on a 2.2-kilobase NcoI-ClaI fragment of genomic DNA. The polypeptide encoded within this DNA fragment was identified following T7 RNA polymerase-dependent induction and estimated to be Mr = 62,000 using SDS-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of acyl-CoA synthetase was determined by automated sequencing to be Met-Lys-Lys-Val-Trp-Leu-Asn-Arg-Tyr-Pro. Sequence analysis of the 2.2-kilobase NcoI-ClaI fragment revealed a single open reading frame encoding these amino acids as the first 10 residues of a protein with a molecular weight of 62,028. The initiation codon for methionine was TTG. Primer extension of total in vivo mRNA from two fadD-specific oligonucleotides defined the transcriptional start at an adenine residue 60 base pairs upstream from the predicted translational start site. Two FadR operator sites of the fadD gene were identified at positions -13 to -29 (OD1) and positions -99 to -115 (OD2) by DNase I footprinting. Comparisons of the predicted amino acid sequence of the E. coli acyl-CoA synthetase to the deduced amino acid sequences of the rat and yeast acyl-CoA synthetases and the firefly luciferase demonstrated that these enzymes shared a significant degree of similarity. Based on the similar reaction mechanisms of these four enzymes, this similarity may define a region required for the same function.

Original languageEnglish (US)
Pages (from-to)25513-25520
Number of pages8
JournalJournal of Biological Chemistry
Volume267
Issue number35
StatePublished - Dec 15 1992

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Coenzyme A Ligases
Cloning
Escherichia coli
Organism Cloning
Genes
Gene Expression
Amino Acids
Amino Acid Sequence
Coenzyme A
Fatty Acids
Acyl-CoA Dehydrogenase
Firefly Luciferases
Initiator Codon
Deoxyribonuclease I
DNA
Adenine
Enterobacteriaceae
Enzymes
Adenosine Monophosphate
Ligases

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cloning, sequencing, and expression of the fadD gene of Escherichia coli encoding acyl coenzyme A synthetase. / Black, Paul N; DiRusso, Concetta C; Metzger, Amy K.; Heimert, Tamra L.

In: Journal of Biological Chemistry, Vol. 267, No. 35, 15.12.1992, p. 25513-25520.

Research output: Contribution to journalArticle

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