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J Bacteriol. Feb 1995; 177(3): 774–782.
PMCID: PMC176656

Cloning, sequence analysis, expression, and inactivation of the Corynebacterium glutamicum icd gene encoding isocitrate dehydrogenase and biochemical characterization of the enzyme.

Abstract

NADP(+)-dependent isocitrate dehydrogenase (ICD) is an important enzyme of the intermediary metabolism, as it controls the carbon flux within the citric acid cycle and supplies the cell with 2-oxoglutarate and NADPH for biosynthetic purposes. In the amino acid-producing organism Corynebacterium glutamicum, the specific activity of ICD was independent of the growth substrate and of the growth phase at approximately 1 U/mg, indicating that this enzyme is constitutively formed. The ICD gene, icd, was isolated, subcloned on a plasmid, and introduced into C. glutamicum. Compared with the wild type, the recombinant strains showed up to 10-fold-higher specific ICD activities. The nucleotide sequence of a 3,595-bp DNA fragment containing the icd gene was determined. The predicted gene product of icd consists of 739 amino acids (M(r) = 80.091) and showed 58.5% identity with the monomeric ICD isozyme II from Vibrio sp. strain ABE-1 but no similarity to any known ICD of the dimeric type. Inactivation of the chromosomal icd gene led to glutamate auxotrophy and to the absence of any detectable ICD activity, suggesting that only a single ICD is present in C. glutamicum. From an icd-overexpressing C. glutamicum strain, ICD was purified and biochemically characterized. The native ICD was found to be a monomer; to be specific for NADP+; to be weakly inhibited by oxaloacetate, 2-oxoglutarate, and citrate; and to be severely inhibited by oxaloacetate plus glyoxylate. The data indicate that ICD from C. glutamicum is structurally similar to ICDs from bacteria of the genera Vibrio, Rhodomicrobium, and Azotobacter but different from all other known procaryotic and eucaryotic ICDs.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.
  • Bennett PM, Holms WH. Reversible inactivation of the isocitrate dehydrogenase of Escherichia coli ML308 during growth on acetate. J Gen Microbiol. 1975 Mar;87(1):37–51. [PubMed]
  • Birnboim HC. A rapid alkaline extraction method for the isolation of plasmid DNA. Methods Enzymol. 1983;100:243–255. [PubMed]
  • Börmann ER, Eikmanns BJ, Sahm H. Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase. Mol Microbiol. 1992 Feb;6(3):317–326. [PubMed]
  • Chung AE, Franzen JS. Oxidized triphosphopyridine nucleotide specific isocitrate dehydrogenase from Azotobacter vinelandii. Isolation and characterization. Biochemistry. 1969 Aug;8(8):3175–3184. [PubMed]
  • Cupp JR, McAlister-Henn L. Cloning and characterization of the gene encoding the IDH1 subunit of NAD(+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae. J Biol Chem. 1992 Aug 15;267(23):16417–16423. [PubMed]
  • Edwards DJ, Heinrikson RL, Chung AE. Triphosphopyridine nucleotide specific isocitrate dehydrogenase from Azotobacter vinelandii. Alkylation of a specific methionine residue and amino acid sequence of the peptide containing this residue. Biochemistry. 1974 Feb 12;13(4):677–683. [PubMed]
  • Eikmanns BJ. Identification, sequence analysis, and expression of a Corynebacterium glutamicum gene cluster encoding the three glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase, 3-phosphoglycerate kinase, and triosephosphate isomerase. J Bacteriol. 1992 Oct;174(19):6076–6086. [PMC free article] [PubMed]
  • Eikmanns BJ, Kleinertz E, Liebl W, Sahm H. A family of Corynebacterium glutamicum/Escherichia coli shuttle vectors for cloning, controlled gene expression, and promoter probing. Gene. 1991 Jun 15;102(1):93–98. [PubMed]
  • Eikmanns BJ, Metzger M, Reinscheid D, Kircher M, Sahm H. Amplification of three threonine biosynthesis genes in Corynebacterium glutamicum and its influence on carbon flux in different strains. Appl Microbiol Biotechnol. 1991 Feb;34(5):617–622. [PubMed]
  • Eikmanns BJ, Thum-Schmitz N, Eggeling L, Lüdtke KU, Sahm H. Nucleotide sequence, expression and transcriptional analysis of the Corynebacterium glutamicum gltA gene encoding citrate synthase. Microbiology. 1994 Aug;140(Pt 8):1817–1828. [PubMed]
  • Fukunaga N, Imagawa S, Sahara T, Ishii A, Suzuki M. Purification and characterization of monomeric isocitrate dehydrogenase with NADP(+)-specificity from Vibrio parahaemolyticus Y-4. J Biochem. 1992 Dec;112(6):849–855. [PubMed]
  • Garnak M, Reeves HC. Phosphorylation of Isocitrate dehydrogenase of Escherichia coli. Science. 1979 Mar 16;203(4385):1111–1112. [PubMed]
  • Haselbeck RJ, Colman RF, McAlister-Henn L. Isolation and sequence of a cDNA encoding porcine mitochondrial NADP-specific isocitrate dehydrogenase. Biochemistry. 1992 Jul 14;31(27):6219–6223. [PubMed]
  • Haselbeck RJ, McAlister-Henn L. Isolation, nucleotide sequence, and disruption of the Saccharomyces cerevisiae gene encoding mitochondrial NADP(H)-specific isocitrate dehydrogenase. J Biol Chem. 1991 Feb 5;266(4):2339–2345. [PubMed]
  • Howard RL, Becker RR. Isolation and some properties of the triphosphopyridine nucleotide isocitrate dehydrogenase from Bacillus stearothermophilus. J Biol Chem. 1970 Jun;245(12):3186–3194. [PubMed]
  • Huh TL, Ryu JH, Huh JW, Sung HC, Oh IU, Song BJ, Veech RL. Cloning of a cDNA encoding bovine mitochondrial NADP(+)-specific isocitrate dehydrogenase and structural comparison with its isoenzymes from different species. Biochem J. 1993 Jun 15;292(Pt 3):705–710. [PMC free article] [PubMed]
  • Hurley JH, Dean AM, Koshland DE, Jr, Stroud RM. Catalytic mechanism of NADP(+)-dependent isocitrate dehydrogenase: implications from the structures of magnesium-isocitrate and NADP+ complexes. Biochemistry. 1991 Sep 3;30(35):8671–8678. [PubMed]
  • Hurley JH, Dean AM, Sohl JL, Koshland DE, Jr, Stroud RM. Regulation of an enzyme by phosphorylation at the active site. Science. 1990 Aug 31;249(4972):1012–1016. [PubMed]
  • Ishii A, Ochiai T, Imagawa S, Fukunaga N, Sasaki S, Minowa O, Mizuno Y, Shiokawa H. Isozymes of isocitrate dehydrogenase from an obligately psychrophilic bacterium, Vibrio sp. strain ABE-1: purification, and modulation of activities by growth conditions. J Biochem. 1987 Dec;102(6):1489–1498. [PubMed]
  • Ishii A, Suzuki M, Sahara T, Takada Y, Sasaki S, Fukunaga N. Genes encoding two isocitrate dehydrogenase isozymes of a psychrophilic bacterium, Vibrio sp. strain ABE-1. J Bacteriol. 1993 Nov;175(21):6873–6880. [PMC free article] [PubMed]
  • Jin S, Sonenshein AL. Identification of two distinct Bacillus subtilis citrate synthase genes. J Bacteriol. 1994 Aug;176(15):4669–4679. [PMC free article] [PubMed]
  • LaPorte DC. The isocitrate dehydrogenase phosphorylation cycle: regulation and enzymology. J Cell Biochem. 1993 Jan;51(1):14–18. [PubMed]
  • LaPorte DC, Thorsness PE, Koshland DE., Jr Compensatory phosphorylation of isocitrate dehydrogenase. A mechanism for adaptation to the intracellular environment. J Biol Chem. 1985 Sep 5;260(19):10563–10568. [PubMed]
  • Leyland ML, Kelly DJ. Purification and characterization of a monomeric isocitrate dehydrogenase with dual coenzyme specificity from the photosynthetic bacterium Rhodomicrobium vannielii. Eur J Biochem. 1991 Nov 15;202(1):85–93. [PubMed]
  • Liebl W, Bayerl A, Schein B, Stillner U, Schleifer KH. High efficiency electroporation of intact Corynebacterium glutamicum cells. FEMS Microbiol Lett. 1989 Dec;53(3):299–303. [PubMed]
  • Miyazaki K, Eguchi H, Yamagishi A, Wakagi T, Oshima T. Molecular cloning of the isocitrate dehydrogenase gene of an extreme thermophile, Thermus thermophilus HB8. Appl Environ Microbiol. 1992 Jan;58(1):93–98. [PMC free article] [PubMed]
  • Muro-Pastor MI, Florencio FJ. NADP(+)-isocitrate dehydrogenase from the cyanobacterium Anabaena sp. strain PCC 7120: purification and characterization of the enzyme and cloning, sequencing, and disruption of the icd gene. J Bacteriol. 1994 May;176(9):2718–2726. [PMC free article] [PubMed]
  • NACHLAS MM, DAVIDSON MB, GOLDBERG JD, SELIGMAN AM. Colorimetric method for the measurement of isocitric dehydrogenase activity. J Lab Clin Med. 1963 Jul;62:148–158. [PubMed]
  • Nimmo HG. Kinetic mechanism of Escherichia coli isocitrate dehydrogenase and its inhibition by glyoxylate and oxaloacetate. Biochem J. 1986 Mar 1;234(2):317–323. [PMC free article] [PubMed]
  • Ozaki H, Shiio I. Regulation of the TCA and glyoxylate cycles in Brevibacterium flavum. I. Ingibition of isocitrate lyase and isocitrate dehydrogenase by organic acids related to the TCA and glyoxylate cycles. J Biochem. 1968 Sep;64(3):355–363. [PubMed]
  • Reinscheid DJ, Eikmanns BJ, Sahm H. Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme. J Bacteriol. 1994 Jun;176(12):3474–3483. [PMC free article] [PubMed]
  • Sanger F, Nicklen S, Coulson AR. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. [PMC free article] [PubMed]
  • Schäfer A, Kalinowski J, Simon R, Seep-Feldhaus AH, Pühler A. High-frequency conjugal plasmid transfer from gram-negative Escherichia coli to various gram-positive coryneform bacteria. J Bacteriol. 1990 Mar;172(3):1663–1666. [PMC free article] [PubMed]
  • Schwarzer A, Pühler A. Manipulation of Corynebacterium glutamicum by gene disruption and replacement. Biotechnology (N Y) 1991 Jan;9(1):84–87. [PubMed]
  • Scrutton NS, Berry A, Perham RN. Redesign of the coenzyme specificity of a dehydrogenase by protein engineering. Nature. 1990 Jan 4;343(6253):38–43. [PubMed]
  • Self CH, Weitzman PD. The isocitrate dehydrogenases of Acinetobacter lwoffi. Separation and properties of two nicotinamide-adenine dinucleotide phosphate-linked isoenzymes. Biochem J. 1972 Nov;130(1):211–219. [PMC free article] [PubMed]
  • Shiio I, Ozaki H. Concerted inhibition of isocitrate dehydrogenase by glyoxylate plus oxalacetate. J Biochem. 1968 Jul;64(1):45–53. [PubMed]
  • Shikata S, Ozaki K, Kawai S, Ito S, Okamoto K. Purification and characterization of NADP+-linked isocitrate dehydrogenase from an alkalophilic Bacillus. Biochim Biophys Acta. 1988 Feb 10;952(3):282–289. [PubMed]
  • Stoddard BL, Koshland DE., Jr Structure of isocitrate dehydrogenase with alpha-ketoglutarate at 2.7-A resolution: conformational changes induced by decarboxylation of isocitrate. Biochemistry. 1993 Sep 14;32(36):9317–9322. [PubMed]
  • Thorsness PE, Koshland DE., Jr Inactivation of isocitrate dehydrogenase by phosphorylation is mediated by the negative charge of the phosphate. J Biol Chem. 1987 Aug 5;262(22):10422–10425. [PubMed]
  • Tinoco I, Jr, Borer PN, Dengler B, Levin MD, Uhlenbeck OC, Crothers DM, Bralla J. Improved estimation of secondary structure in ribonucleic acids. Nat New Biol. 1973 Nov 14;246(150):40–41. [PubMed]
  • Udvardi MK, McDermott TR, Kahn ML. Isolation and characterization of a cDNA encoding NADP(+)-specific isocitrate dehydrogenase from soybean (Glycine max). Plant Mol Biol. 1993 Mar;21(5):739–752. [PubMed]
  • Vanni P, Giachetti E, Pinzauti G, McFadden BA. Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp Biochem Physiol B. 1990;95(3):431–458. [PubMed]
  • Vieira J, Messing J. The pUC plasmids, an M13mp7-derived system for insertion mutagenesis and sequencing with synthetic universal primers. Gene. 1982 Oct;19(3):259–268. [PubMed]
  • Walsh K, Koshland DE., Jr Branch point control by the phosphorylation state of isocitrate dehydrogenase. A quantitative examination of fluxes during a regulatory transition. J Biol Chem. 1985 Jul 15;260(14):8430–8437. [PubMed]

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