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Title
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Cloning and expression of the fadH gene and characterization of the gene product 2,4-dienoyl coenzyme A reductase from Escherichia coli.
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Authors
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X.Y.He,
S.Y.Yang,
H.Schulz.
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Ref.
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Eur J Biochem, 1997,
248,
516-520.
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PubMed id
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Abstract
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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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