Studies of human mitochondrial 2,4-dienoyl-CoA reductase.
Mitochondrial 2,4-dienoyl-CoA reductase is a key enzyme for the beta-oxidation of unsaturated fatty acids. Sequence alignment indicates that there are five highly conserved acidic residues, one of which might act as a proton donor. We constructed five mutant expression plasmids of human mitochondrial 2,4-dienoyl-CoA reductase using site-directed mutagenesis. Mutant proteins were overexpressed in Escherichia coli and purified with a nickel metal affinity column. Studies of these mutant proteins were carried out, and the proton donor is likely to be E276. Three substrate analogs were synthesized and characterized. Two analogs, 2-fluoro-2,4-octadienoyl-CoA and 5-methyl-2,4-hexadienoyl-CoA, were substrates of the enzyme. Another analog, 3-furan-2-yl-acrylyl-CoA, was not a substrate, but a competitive inhibitor of the enzyme. These studies increased our understanding of human mitochondrial 2,4-dienoyl-CoA reductase.
Yu
Wenhua
W
Department of Biology and Chemistry, City University of Hong Kong, 83 Tat Chee Avenue, Kowloon, Hong Kong SAR, PR China.
Chu
Xiusheng
X
Chen
Gong
G
Li
Ding
D
D000595:Amino Acid Sequence; D001483:Base Sequence; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005231:Fatty Acids, Unsaturated / Q000138:chemical synthesis / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D044925:Oxidoreductases Acting on CH-CH Group Donors / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism*; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D005231:Fatty Acids, Unsaturated; D011994:Recombinant Proteins; D004247:DNA; D044925:Oxidoreductases Acting on CH-CH Group Donors; C100153:2,4-dienoyl-CoA reductase