Skip to main content
NCBI home page
Journal of Bacteriology logoLink to Journal of Bacteriology
. 1994 Jun;176(12):3474–3483. doi: 10.1128/jb.176.12.3474-3483.1994

Characterization of the isocitrate lyase gene from Corynebacterium glutamicum and biochemical analysis of the enzyme.

D J Reinscheid 1, B J Eikmanns 1, H Sahm 1
PMCID: PMC205534  PMID: 8206824

Abstract

Isocitrate lyase is a key enzyme in the glyoxylate cycle and is essential as an anaplerotic enzyme for growth on acetate as a carbon source. It is assumed to be of major importance in carbon flux control in the amino acid-producing organism Corynebacterium glutamicum. In crude extracts of C. glutamicum, the specific activities of isocitrate lyase were found to be 0.01 U/mg of protein after growth on glucose and 2.8 U/mg of protein after growth on acetate, indicating tight regulation. The isocitrate lyase gene, aceA, was isolated, subcloned, and characterized. The predicted gene product of aceA consists of 432 amino acids (M(r), 47,228) and shows up to 57% identity to the respective enzymes from other organisms. Downstream of aceA, a gene essential for thiamine biosynthesis was identified. Overexpression of aceA in C. glutamicum resulted in specific activities of 0.1 and 7.4 U/mg of protein in minimal medium containing glucose and acetate, respectively. Inactivation of the chromosomal aceA gene led to an inability to grow on acetate and to the absence of any detectable isocitrate lyase activity. Isocitrate lyase was purified to apparent homogeneity and subjected to biochemical analysis. The native enzyme was shown to be a tetramer of identical subunits, to exhibit an ordered Uni-Bi mechanism of catalysis, and to be effectively inhibited by 3-phosphoglycerate, 6-phosphogluconate, phosphoenolpyruvate, fructose-1,6-bisphosphate, and succinate.

Full text

PDF
3474

Images in this article

3480Image
on p.3480

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Atomi H., Ueda M., Hikida M., Hishida T., Teranishi Y., Tanaka A. Peroxisomal isocitrate lyase of the n-alkane-assimilating yeast Candida tropicalis: gene analysis and characterization. J Biochem. 1990 Feb;107(2):262–266. doi: 10.1093/oxfordjournals.jbchem.a123036. [DOI] [PubMed] [Google Scholar]
  2. Barth G., Scheuber T. Cloning of the isocitrate lyase gene (ICL1) from Yarrowia lipolytica and characterization of the deduced protein. Mol Gen Genet. 1993 Nov;241(3-4):422–430. doi: 10.1007/BF00284696. [DOI] [PubMed] [Google Scholar]
  3. Behari R., Baker A. The carboxyl terminus of isocitrate lyase is not essential for import into glyoxysomes in an in vitro system. J Biol Chem. 1993 Apr 5;268(10):7315–7322. [PubMed] [Google Scholar]
  4. Börmann E. R., Eikmanns B. J., Sahm H. Molecular analysis of the Corynebacterium glutamicum gdh gene encoding glutamate dehydrogenase. Mol Microbiol. 1992 Feb;6(3):317–326. doi: 10.1111/j.1365-2958.1992.tb01474.x. [DOI] [PubMed] [Google Scholar]
  5. Chell R. M., Sundaram T. K., Wilkinson A. E. Isolation and characterization of isocitrate lyase from a thermophilic Bacillus sp. Biochem J. 1978 Jul 1;173(1):165–177. doi: 10.1042/bj1730165. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Conder M. J., Ko Y. H., McFadden B. A. Purification of isocitrate lyase from Escherichia coli and watermelon using fast protein liquid chromatography. Prep Biochem. 1988;18(4):431–442. doi: 10.1080/00327488808062542. [DOI] [PubMed] [Google Scholar]
  7. Diehl P., McFadden B. A. Site-directed mutagenesis of lysine 193 in Escherichia coli isocitrate lyase by use of unique restriction enzyme site elimination. J Bacteriol. 1993 Apr;175(8):2263–2270. doi: 10.1128/jb.175.8.2263-2270.1993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Eikmanns B. J. 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. doi: 10.1128/jb.174.19.6076-6086.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Eikmanns B. J., 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. doi: 10.1016/0378-1119(91)90545-m. [DOI] [PubMed] [Google Scholar]
  10. Eikmanns B. J., 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. doi: 10.1007/BF00167910. [DOI] [PubMed] [Google Scholar]
  11. Fernández E., Moreno F., Rodicio R. The ICL1 gene from Saccharomyces cerevisiae. Eur J Biochem. 1992 Mar 15;204(3):983–990. doi: 10.1111/j.1432-1033.1992.tb16720.x. [DOI] [PubMed] [Google Scholar]
  12. Gainey L. D., Connerton I. F., Lewis E. H., Turner G., Ballance D. J. Characterization of the glyoxysomal isocitrate lyase genes of Aspergillus nidulans (acuD) and Neurospora crassa (acu-3). Curr Genet. 1992 Jan;21(1):43–47. doi: 10.1007/BF00318653. [DOI] [PubMed] [Google Scholar]
  13. Hillier S., Charnetzky W. T. Glyoxylate bypass enzymes in Yersinia species and multiple forms of isocitrate lyase in Yersinia pestis. J Bacteriol. 1981 Jan;145(1):452–458. doi: 10.1128/jb.145.1.452-458.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Hohn B., Collins J. A small cosmid for efficient cloning of large DNA fragments. Gene. 1980 Nov;11(3-4):291–298. doi: 10.1016/0378-1119(80)90069-4. [DOI] [PubMed] [Google Scholar]
  15. Hoyt J. C., Johnson K. E., Reeves H. C. Purification and characterization of Acinetobacter calcoaceticus isocitrate lyase. J Bacteriol. 1991 Nov;173(21):6844–6848. doi: 10.1128/jb.173.21.6844-6848.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Ko Y. H., Cremo C. R., McFadden B. A. Vanadate-dependent photomodification of serine 319 and 321 in the active site of isocitrate lyase from Escherichia coli. J Biol Chem. 1992 Jan 5;267(1):91–95. [PubMed] [Google Scholar]
  17. Ko Y. H., McFadden B. A. Alkylation of isocitrate lyase from Escherichia coli by 3-bromopyruvate. Arch Biochem Biophys. 1990 May 1;278(2):373–380. doi: 10.1016/0003-9861(90)90273-2. [DOI] [PubMed] [Google Scholar]
  18. Kornberg H. L. The role and control of the glyoxylate cycle in Escherichia coli. Biochem J. 1966 Apr;99(1):1–11. doi: 10.1042/bj0990001. [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. LaPorte D. C., Koshland D. E., Jr Phosphorylation of isocitrate dehydrogenase as a demonstration of enhanced sensitivity in covalent regulation. Nature. 1983 Sep 22;305(5932):286–290. doi: 10.1038/305286a0. [DOI] [PubMed] [Google Scholar]
  20. LaPorte D. C., Thorsness P. E., Koshland D. E., 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] [Google Scholar]
  21. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  22. Liebl W., Bayerl A., Schein B., Stillner U., Schleifer K. H. High efficiency electroporation of intact Corynebacterium glutamicum cells. FEMS Microbiol Lett. 1989 Dec;53(3):299–303. doi: 10.1016/0378-1097(89)90234-6. [DOI] [PubMed] [Google Scholar]
  23. MacKintosh C., Nimmo H. G. Purification and regulatory properties of isocitrate lyase from Escherichia coli ML308. Biochem J. 1988 Feb 15;250(1):25–31. doi: 10.1042/bj2500025. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Maloy S. R., Nunn W. D. Genetic regulation of the glyoxylate shunt in Escherichia coli K-12. J Bacteriol. 1982 Jan;149(1):173–180. doi: 10.1128/jb.149.1.173-180.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Matsuoka M., McFadden B. A. Isolation, hyperexpression, and sequencing of the aceA gene encoding isocitrate lyase in Escherichia coli. J Bacteriol. 1988 Oct;170(10):4528–4536. doi: 10.1128/jb.170.10.4528-4536.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Nimmo H. G., Douglas F., Kleanthous C., Campbell D. G., MacKintosh C. Identification of a cysteine residue at the active site of Escherichia coli isocitrate lyase. Biochem J. 1989 Jul 15;261(2):431–435. doi: 10.1042/bj2610431. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. 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. doi: 10.1093/oxfordjournals.jbchem.a128902. [DOI] [PubMed] [Google Scholar]
  28. Rieul C., Bleicher F., Duclos B., Cortay J. C., Cozzone A. J. Nucleotide sequence of the aceA gene coding for isocitrate lyase in Escherichia coli. Nucleic Acids Res. 1988 Jun 24;16(12):5689–5689. doi: 10.1093/nar/16.12.5689. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Robertson E. F., Hoyt J. C., Reeves H. C. Evidence of histidine phosphorylation in isocitrate lyase from Escherichia coli. J Biol Chem. 1988 Feb 15;263(5):2477–2482. [PubMed] [Google Scholar]
  30. Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Schwarzer A., Pühler A. Manipulation of Corynebacterium glutamicum by gene disruption and replacement. Biotechnology (N Y) 1991 Jan;9(1):84–87. doi: 10.1038/nbt0191-84. [DOI] [PubMed] [Google Scholar]
  32. Vanni P., Giachetti E., Pinzauti G., McFadden B. A. Comparative structure, function and regulation of isocitrate lyase, an important assimilatory enzyme. Comp Biochem Physiol B. 1990;95(3):431–458. doi: 10.1016/0305-0491(90)90002-b. [DOI] [PubMed] [Google Scholar]
  33. 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. doi: 10.1016/0378-1119(82)90015-4. [DOI] [PubMed] [Google Scholar]
  34. Vinopal R. T., Fraenkel D. G. Phenotypic suppression of phosphofructokinase mutations in Escherichia coli by constitutive expression of the glyoxylate shunt. J Bacteriol. 1974 Jun;118(3):1090–1100. doi: 10.1128/jb.118.3.1090-1100.1974. [DOI] [PMC free article] [PubMed] [Google Scholar]
  35. Walsh K., Koshland D. E., 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] [Google Scholar]
  36. Warren W. A. Catalysis of both oxidation and reduction of glyoxylate by pig heart lactate dehydrogenase isozyme 1. J Biol Chem. 1970 Apr 10;245(7):1675–1681. [PubMed] [Google Scholar]
  37. Wilson R. B., Maloy S. R. Isolation and characterization of Salmonella typhimurium glyoxylate shunt mutants. J Bacteriol. 1987 Jul;169(7):3029–3034. doi: 10.1128/jb.169.7.3029-3034.1987. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Zhang J. Z., Gomez-Pedrozo M., Baden C. S., Harada J. J. Two classes of isocitrate lyase genes are expressed during late embryogeny and postgermination in Brassica napus L. Mol Gen Genet. 1993 Apr;238(1-2):177–184. doi: 10.1007/BF00279545. [DOI] [PubMed] [Google Scholar]

Articles from Journal of Bacteriology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES