NAD(+)-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae.
NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae is composed of two nonidentical subunits, designated IDH1 (Mr approximately 40,000) and IDH2 (Mr approximately 39,000). We have isolated and characterized a yeast genomic clone containing the IDH2 gene. The amino acid sequence deduced from the gene indicates that IDH2 is synthesized as a precursor of 369 amino acids (Mr 39,694) and is processed upon mitochondrial import to yield a mature protein of 354 amino acids (Mr 37,755). Amino acid sequence comparison between S. cerevisiae IDH2 and S. cerevisiae NADP(+)-dependent isocitrate dehydrogenase shows no significant sequence identity, whereas comparison of IDH2 and Escherichia coli NADP(+)-dependent isocitrate dehydrogenase reveals a 33% sequence identity. To confirm the identity of the IDH2 gene and examine the relationship between IDH1 and IDH2, the IDH2 gene was disrupted by genomic replacement in a haploid yeast strain. The disruption strain expressed no detectable IDH2, as determined by Western blot analysis, and was found to lack NAD(+)-dependent isocitrate dehydrogenase activity, indicating that IDH2 is essential for a functional enzyme. Overexpression of IDH2, however, did not result in increased NAD(+)-dependent isocitrate dehydrogenase activity, suggesting that both IDH1 and IDH2 subunits are required for catalytic activity. The disruption strain was unable to utilize acetate as a carbon source and exhibited a 2-fold slower growth rate than wild type strains on glycerol or lactate. This growth phenotype is consistent with NAD(+)-dependent isocitrate dehydrogenase performing an essential role in the oxidative function of the citric acid cycle.
Cupp
J R
JR
Department of Biological Chemistry, University of California, Irvine 92717.
McAlister-Henn
L
L
D000595:Amino Acid Sequence; D001483:Base Sequence; D015139:Blotting, Southern; D003001:Cloning, Molecular; D004271:DNA, Fungal / Q000235:genetics / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D005800:Genes, Fungal; D007521:Isocitrate Dehydrogenase / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D007527:Isoenzymes / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008928:Mitochondria / Q000201:enzymology; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009243:NAD / Q000378:metabolism; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D015183:Restriction Mapping; D012441:Saccharomyces cerevisiae / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D012689:Sequence Homology, Nucleic Acid
D004271:DNA, Fungal; D007527:Isoenzymes; D011994:Recombinant Proteins; D009243:NAD; D007521:Isocitrate Dehydrogenase