Cloning and expression of the fadH gene and characterization of the gene product 2,4-dienoyl coenzyme A reductase from Escherichia coli.
The fadH gene coding for an NADPH-dependent 2.4-dienoyl-CoA reductase from Escherichia coli has been cloned by the polymerase chain reaction. This gene is located at 67.65 min on the E. coli chromosome. The complete open reading frame contains 2019 bp coding for the processed protein of 671 amino acid residues, with a calculated molecular mass of 72.55 kDa, which lacks the N-terminal methionine. Construction and expression of the plasmid pNDH, which contained the fadH gene under the control of the T7 promoter, resulted in a 110-fold increase in the reductase activity above the level detected in E. coli cells containing the control vector. The kinetic parameters of the purified reductase were determined to be 50 microM and 2.3 microM for the Km values of NADPH and 2-trans, 4-trans-decadienoyl-CoA, respectively, and 16 s(-1) for the k(cat) value. Analysis of the kinetic data revealed that the reaction catalyzed by this enzyme proceeds via a ping-pong mechanism. The observed dissimilarity between the E. coli and mammalian 2,4-dienoyl-CoA reductase sequences suggests that they have evolved from distinct ancestral genes. Sequence analysis also suggests that the N-terminal part of the E. coli reductase contains the FAD-binding domain whereas the NADPH-binding domain is located in the C-terminal region of the protein.
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Department of Chemistry, City College of the City University of New York, New York 10031, USA.
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Schulz
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D000595:Amino Acid Sequence; D001483:Base Sequence; D002874:Chromosome Mapping; D002876:Chromosomes, Bacterial; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics*; D044943:Fatty Acid Desaturases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D008969:Molecular Sequence Data; D044925:Oxidoreductases Acting on CH-CH Group Donors; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C100153:2,4-dienoyl-CoA reductase