Identification of the active site catalytic residue in human isovaleryl-CoA dehydrogenase.
Isovaleryl-CoA dehydrogenase (IVD) is a homotetrameric flavoenzyme which catalyzes the conversion of isovaleryl-CoA to 3-methylcrotonyl-CoA. E376 of pig medium chain acyl-CoA dehydrogenase (MCAD), a homologous enzyme, has been identified as the active site catalytic residue. Amino acid sequence alignment shows that A375 is the corresponding residue in human IVD. Using the atomic coordinates determined for MCAD, molecular modeling suggests that E254 is the substituting catalytic residue in IVD. To substantiate the importance of this residue for enzyme function, cDNAs for the wild-type human IVD and E254G, E254D, E254Q, and E254G/A375E mutant IVDs were constructed and cloned into a prokaryotic expression vector. The proteins were synthesized in Escherichia coli and purified, and their properties were examined. The catalytic activity of the recombinant wild-type IVD was the highest in the presence of isovaleryl-CoA, and its UV/visible light spectrum in the presence of isovaleryl-CoA showed quenching of its characteristic absorption in the 445-nm region and appearance of absorption at 600 nm. The E254G and E254Q mutant IVDs had no detectable enzymatic activity, and isovaleryl-CoA did not induce quenching of the absorption in the 445-nm region or the appearance of absorption at 600 nm. The E254D mutant IVD had residual activity for isovaleryl-CoA, and its spectrum was altered compared to that of the wild type. The E254G/A375E mutant IVD exhibited catalytic activity toward isovaleryl-CoA, and its spectrum in the absence or presence of the substrate was similar to that of the wild-type IVD.(ABSTRACT TRUNCATED AT 250 WORDS)
Mohsen
A W
AW
Department of Medical Genetics, Mayo Clinic, Rochester, Minnesota 55905, USA.
Vockley
J
J
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002384:Catalysis; D017931:DNA Primers; D006801:Humans; D050769:Isovaleryl-CoA Dehydrogenase; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D044925:Oxidoreductases Acting on CH-CH Group Donors; D013057:Spectrum Analysis; D013552:Swine
D017931:DNA Primers; D010088:Oxidoreductases; D044925:Oxidoreductases Acting on CH-CH Group Donors; D050769:Isovaleryl-CoA Dehydrogenase