Identification of Caenorhabditis elegans isovaleryl-CoA dehydrogenase and structural comparison with other acyl-CoA dehydrogenases.
Isovaleryl-CoA dehydrogenase (IVD) is a flavoenzyme, which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA in the leucine catabolism pathway and transfers electrons to the electron-transferring flavoprotein (ETF). IVDs from human and rat have been identified and characterized previously. In this study, the gene coding for Caenorhabditis elegans IVD has been identified from a published cDNA sequence and molecular modeling has been performed using the human IVD atomic coordinates. The coding sequence for the mature form of the enzyme was expressed in Escherichia coli, and the recombinant nematode IVD enzyme was purified to essential homogeneity. Its spectrum is typical of recombinant FAD-containing acyl-CoA dehydrogenases and shows a minor broad absorption band at 650-700 nm characteristic of an IVD:CoA persulfide charge-transfer complex. Following treatment of the enzyme with sodium dithionite to remove the bound CoA persulfide, the K(m) values for isovaleryl-, butyryl-, valeryl-, and hexanoyl-CoA were estimated to be 2.5, 36.2, 10.5, and 33.8 microM, respectively, using the ETF fluorescence reduction assay. The catalytic efficiency (k(cat)/K(m)) for these substrates was 56.9, 1.3, 13.7, and 3.2 microM(-1). min(-1) per mole of FAD, respectively. The apparent binding constant (K(D app)) of the recombinant IVD determined spectrally for isovaleryl-CoA was 0.34 microM. These kinetic parameters confirm that isovaleryl-CoA is the preferred substrate for the purified enzyme. The variability in the protein structure among known and putative IVDs from various species is discussed in the context of possible mechanisms for modulating enzyme activity.
Mohsen
A W
AW
Department of Medical Genetics, Mayo Clinic and Mayo Foundation, Rochester, Minnesota 55905, USA.
Navarette
B
B
Vockley
J
J
D000214:Acyl Coenzyme A / Q000378:metabolism; D042964:Acyl-CoA Dehydrogenase; D000595:Amino Acid Sequence; D000818:Animals; D017173:Caenorhabditis elegans / Q000201:enzymology / Q000235:genetics*; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D044943:Fatty Acid Desaturases / Q000235:genetics*; D050769:Isovaleryl-CoA Dehydrogenase; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D044925:Oxidoreductases Acting on CH-CH Group Donors; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013053:Spectrophotometry; D013379:Substrate Specificity
D000214:Acyl Coenzyme A; D018076:DNA, Complementary; D010088:Oxidoreductases; D044943:Fatty Acid Desaturases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050769:Isovaleryl-CoA Dehydrogenase; D042964:Acyl-CoA Dehydrogenase