NAD+-dependent isocitrate dehydrogenase. Cloning, nucleotide sequence, and disruption of the IDH2 gene from Saccharomyces cerevisiae

• Published in: The Journal of biological chemistry Vol. 266; no. 33; pp. 22199 - 22205
• Main Authors: Cupp, J.R. (University of California, Irvine, CA), McAlister-Henn, L
• Format: Journal Article
• Language: English
• Published: Bethesda, MD American Society for Biochemistry and Molecular Biology 25-11-1991
• Subjects: Amino Acid Sequence; Analytical, structural and metabolic biochemistry; Base Sequence; Biological and medical sciences; Blotting, Southern; CLONACION; CLONAGE; Cloning, Molecular; DNA, Fungal - genetics; DNA, Fungal - isolation & purification; Enzymes and enzyme inhibitors; Escherichia coli - enzymology; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; GENE; GENES; Genes, Fungal; Isocitrate Dehydrogenase - genetics; Isocitrate Dehydrogenase - isolation & purification; Isocitrate Dehydrogenase - metabolism; ISOCITRATE DESHYDROGENASE; ISOCITRATO DESHIDROGENASA; Isoenzymes - genetics; Isoenzymes - isolation & purification; Isoenzymes - metabolism; Mitochondria - enzymology; Molecular Sequence Data; Molecular Weight; NAD - metabolism; NUCLEOTIDE; NUCLEOTIDOS; Oxidoreductases; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Restriction Mapping; SACCHAROMYCES CEREVISIAE; Saccharomyces cerevisiae - enzymology; Saccharomyces cerevisiae - genetics; Saccharomyces cerevisiae - growth & development; Sequence Homology, Nucleic Acid; Fungi; Yeast; Nucleotide sequence; Enzyme; Restriction map; Isolation; Ascomycetes; Molecular cloning; Isocitrate dehydrogenase (NAD+); Saccharomyces cerevisiae; Aminoacid sequence; Thallophyta
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/s0021-9258(18)54554-3

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