Cloning, expression, and purification of the functional 2,4-dienoyl-CoA reductase from rat liver mitochondria.
The mitochondrial 2,4-dienoyl-CoA reductase (EC 1.3.1.34) is an auxiliary enzyme for the beta-oxidation of unsaturated fatty acids. Import of this enzyme into the mitochondria requires a mitochondrial signal sequence at the amino terminus of the polypeptide chain which is processed/removed once inside the mitochondria. The cDNA of the full-length 2,4-dienoyl-CoA reductase was previously cloned as pRDR181. PCR methodologies were used to subclone the gene encoding the functional 2,4-dienoyl-CoA reductase from pRDR181. The PCR product was inserted into a pET15b expression vector and overexpressed in Escherichia coli. The soluble expressed protein can be separated into high- and low-activity fractions. The low-activity fraction can be converted to the high specific activity form by thermal annealing, suggesting it is a metastable misfolded form of the enzyme. Using ion-exchange and affinity chromatography, the enzyme has been purified to homogeneity and exhibits a single band on Coomassie blue-stained SDS-PAGE. The molecular mass of 32,413 Da determined by electrospray ionization-mass spectrometry indicates that the amino-terminal methionine had been removed. The Michaelis constants for trans-2, trans-4-hexadienoyl-CoA and NADPH were determined to be 0.46 and 2.5 microM, respectively; a turnover number of 2.1 s(-1) was calculated.
Fillgrove
K L
KL
Department of Biochemistry, Case Western Reserve University, Cleveland, Ohio 44106-4935, USA.
Anderson
V E
VE
Mizugaki
M
M
D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D017931:DNA Primers / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000235:genetics* / Q000302:isolation & purification* / Q000378:metabolism; D015870:Gene Expression; D066298:In Vitro Techniques; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D044925:Oxidoreductases Acting on CH-CH Group Donors; D016133:Polymerase Chain Reaction; D051381:Rats; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D017931:DNA Primers; D011994:Recombinant Proteins; D044943:Fatty Acid Desaturases; D044925:Oxidoreductases Acting on CH-CH Group Donors; C100153:2,4-dienoyl-CoA reductase