AcnC of is a 2-methylcitrate dehydratase (PrpD) that can use citrate and isocitrate as substrates Free

Abstract

possesses two well-characterized aconitases (AcnA and AcnB) and a minor activity (designated AcnC) that is retained by double mutants and represents no more than 5% of total wild-type aconitase activity. Here it is shown that a 2-methylcitrate dehydratase (PrpD) encoded by the gene of the propionate catabolic operon () is identical to AcnC. Inactivation of abolished the residual aconitase activity of an AcnAB-null strain, whereas inactivation of , an unidentified paralogue, had no significant effect on AcnC activity. Purified PrpD catalysed the dehydration of citrate and isocitrate but was most active with 2-methylcitrate. PrpD also catalysed the dehydration of several other hydroxy acids but failed to hydrate -aconitate and related substrates containing double bonds, indicating that PrpD is not a typical aconitase but a dehydratase. Purified PrpD was shown to be a monomeric iron–sulphur protein ( 54000) having one unstable [2Fe–2S] cluster per monomer, which is needed for maximum catalytic activity and can be reconstituted by treatment with Fe under reducing conditions.

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2002-01-01
2024-03-28
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References

  1. Alen C., Sonenshein A. L. 1999; Bacillus subtilis aconitase is an RNA-binding protein. Proc Natl Acad Sci USA 96:10412–10417 [CrossRef]
    [Google Scholar]
  2. Beinert H. 1983; Semi-micro methods for analysis of labile sulfur and of labile sulfide plus sulfane sulfur in unusually stable iron–sulfur proteins. Anal Biochem 131:373–378 [CrossRef]
    [Google Scholar]
  3. Beinert H., Kennedy M. C., Stout C. D. 1996; Aconitase as iron–sulfur protein, enzyme and iron-regulatory protein. Chem Rev 96:2335–2373 [CrossRef]
    [Google Scholar]
  4. Bell P. J., Andrews S. C., Sivak M. N., Guest J. R. 1989; Nucleotide sequence of the FNR-regulated fumarase gene ( fumB ) of Escherichia coli K-12. J Bacteriol 171:3494–3503
    [Google Scholar]
  5. Bennett B., Gruer M. J., Guest J. R., Thomson A. J. 1995; Spectroscopic characterisation of an aconitase (AcnA) of Escherichia coli . Eur J Biochem 233:317–326 [CrossRef]
    [Google Scholar]
  6. Blattner F. R., Bloch C. A. 14 other authors Plunkett G. III 1997; The complete genome sequence of Escherichia coli K-12. Science 277:1453–1470 [CrossRef]
    [Google Scholar]
  7. Bradbury A. J., Gruer M. J., Rudd K. E., Guest J. R. 1996; The second aconitase (AcnB) of Escherichia coli. . Microbiology 142:389–400 [CrossRef]
    [Google Scholar]
  8. Brock M., Fischer R., Linder D., Buckel W. 2000; Methylcitrate synthase from Aspergillus nidulans : implications for propionate as an antifungal agent. Mol Microbiol 35:961–973 [CrossRef]
    [Google Scholar]
  9. Brock M., Darley D., Textor S., Buckel W. 2001; 2-Methylisocitrate lyases from the bacterium Escherichia coli and the filamentous fungus Aspergillus nidulans . Characterization and comparison of both enzymes. Eur J Biochem 268:3577–3586 [CrossRef]
    [Google Scholar]
  10. Chang A. C. Y., Cohen S. N. 1978; Construction and characterization of amplifiable multicopy DNA cloning vehicles derived from the P15A cryptic miniplasmid. J Bacteriol 134:1141–1156
    [Google Scholar]
  11. Cole S. T., Guest J. R. 1980; Genetic and physical characterisation of lambda transducing phages (λ frdA ) containing the fumarate reductase gene of Escherichia coli K12. Mol Gen Genet 178:409–418 [CrossRef]
    [Google Scholar]
  12. Cunningham L., Gruer M. J., Guest J. R. 1997; Transcriptional regulation of the aconitase genes ( acnA and acnB ) of Escherichia coli . Microbiology 143:3795–3805 [CrossRef]
    [Google Scholar]
  13. Flint D. H., Emptage M. H. 1988; Dihydroxy acid dehydratase from spinach contains a [2Fe–2S] cluster. J Biol Chem 263:3558–3564
    [Google Scholar]
  14. Flint D. H., Emptage M. H., Guest J. R. 1992; Fumarase A from Escherichia coli : purification and characterization as an iron–sulfur cluster containing enzyme. Biochemistry 31:10331–10337 [CrossRef]
    [Google Scholar]
  15. Gerike U., Hough D. W., Russell N. J., Dyall-Smith M. L., Danson M. J. 1998; Citrate synthase and 2-methylcitrate synthase: structural, functional and evolutionary relationships. Microbiology 144:929–935 [CrossRef]
    [Google Scholar]
  16. Green J., Bennett B., Jordan P., Ralph E. T., Thomson A. J., Guest J. R. 1996; Reconstitution of the [4Fe–4S] cluster in FNR and demonstration of the aerobic–anaerobic transcription switch in vitro . Biochem J 316:887–892
    [Google Scholar]
  17. Gruer M. J., Guest J. R. 1994; Two genetically-distinct and differentially-regulated aconitases (AcnA and AcnB) in Escherichia coli . Microbiology 140:2531–2541 [CrossRef]
    [Google Scholar]
  18. Gruer M. J., Artymiuk P. J., Guest J. R. 1997a; The aconitase family: three structural variations on a common theme. Trends Biochem Sci 22:3–6 [CrossRef]
    [Google Scholar]
  19. Gruer M. J., Bradbury A. J., Guest J. R. 1997b; Construction and properties of aconitase mutants of Escherichia coli . Microbiology 143:1837–1846 [CrossRef]
    [Google Scholar]
  20. Hentze M. W., Kuhn L. C. 1996; Molecular control of vertebrate iron metabolism: mRNA-based regulatory circuits operated by iron, nitric oxide and oxidative stress. Proc Natl Acad Sci USA 93:8175–8182 [CrossRef]
    [Google Scholar]
  21. Horswill A. R., Escalante-Semerena J. C. 1997; Propionate catabolism in Salmonella typhimurium LT2: two divergently transcribed units comprise the prp locus at 8·5 centisomes, prpR encodes a member of the sigma-54 family of activators, and the prpBCDE genes constitute an operon. J Bacteriol 179:928–940
    [Google Scholar]
  22. Horswill A. R., Escalante-Semerena J. C. 1999a; Salmonella typhimurium LT2 catabolizes propionate via the 2-methylcitric acid cycle. J Bacteriol 181:5615–5623
    [Google Scholar]
  23. Horswill A. R., Escalante-Semerena J. C. 1999b; The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase. Microbiology 145:1381–1388 [CrossRef]
    [Google Scholar]
  24. Horswill A. R., Escalante-Semerena J. C. 2001; In vitro conversion of propionate to pyruvate by Salmonella enterica enzymes: 2-methylcitrate dehydratase (PrpD) and aconitase enzymes catalyse the conversion of 2-methylcitrate to 2-methylisocitrate. Biochemistry 40:4703–4713 [CrossRef]
    [Google Scholar]
  25. Jordan P. A., Tang Y., Bradbury A. J., Thomson A. J., Guest J. R. 1999; Biochemical and spectroscopic characterisation of Escherichia coli aconitases (AcnA and AcnB). Biochem J 344:739–746 [CrossRef]
    [Google Scholar]
  26. Kennedy M. C., Emptage M. H., Dreyer J.-L., Beinert H. 1983; The role of iron in the activation–inactivation of aconitase. J Biol Chem 258:11098–11105
    [Google Scholar]
  27. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 277:680–685
    [Google Scholar]
  28. Lakshmi T. M., Helling R. B. 1976; Selection for citrate synthase deficiency in icd mutants of Escherichia coli . J Bacteriol 127:76–83
    [Google Scholar]
  29. Lennox E. S. 1955; Transduction of linked genetic characters of host by bacteriophage P1. Virology 1:190–206 [CrossRef]
    [Google Scholar]
  30. Lauble H., Kennedy M. C., Beinert H., Stout C. D. 1992; Crystal structures of aconitases with isocitrate and nitroisocitrate bound. Biochemistry 31:2735–2748 [CrossRef]
    [Google Scholar]
  31. Marsh P. 1986; ptac-85, an Escherichia coli vector for expression of non-fusion proteins. Nucleic Acids Res 14:3603 [CrossRef]
    [Google Scholar]
  32. Miles J. M., Guest J. R. 1984; Complete nucleotide sequence of the fumarase gene fumA of Escherichia coli . Nucleic Acids Res 12:3631–3642 [CrossRef]
    [Google Scholar]
  33. Prodromou C., Artymiuk P. J., Guest J. R. 1992; The aconitase of Escherichia coli . Eur J Biochem 204:599–609 [CrossRef]
    [Google Scholar]
  34. Robbins A. H., Stout C. D. 1989; The structure of aconitase. Proteins 5:289–312 [CrossRef]
    [Google Scholar]
  35. Sambrook J., Fritsch E. F., Maniatis T. 1989 Molecular Cloning: a Laboratory Manual , 2nd edn. Cold Spring Harbor, NY: Cold Spring Harbor Laboratory;
    [Google Scholar]
  36. Shimada K., Weisberg R. A., Gottesman M. E. 1973; Prophage lambda at unusual chromosomal locations. II. Mutations induced by bacteriophage lambda in Escherichia coli K12. J Mol Biol 80:297–314 [CrossRef]
    [Google Scholar]
  37. Somerville G., Miloryak C. A., Reitzer L. 1999; Physiological characterization of Pseudomonas aeruginosa during exotoxin A synthesis: glutamate, iron limitation, and aconitase activity. J Bacteriol 181:1072–1078
    [Google Scholar]
  38. Tang Y., Guest J. R. 1999; Direct evidence for mRNA binding and post-transcriptional regulation by Escherichia coli aconitases. Microbiology 145:3069–3079
    [Google Scholar]
  39. Textor S., Wendisch V. F., De Graaf A. A., Linder M. I., Linder D., Buckel W., Müller U. 1997; Propionate oxidation in E. coli: evidence for operation of a methylcitrate cycle in bacteria. Arch Microbiol 168:428–436 [CrossRef]
    [Google Scholar]
  40. VanRooyen J. P. G., Mienie J., Erasmus E., DeWet W. J., Ketting D., Duran M., Wadman S. K. 1994; Identification of the stereoisomeric configurations of methylcitric acid produced by si -citrate synthase and methylcitrate synthase using capillary gas-chromatography mass-spectrometry. J Inherit Metab Dis 17:738–747 [CrossRef]
    [Google Scholar]
  41. Wilde R. J., Jeyaseelan K., Guest J. R. 1986; Cloning of the aconitase gene ( acn ) of Escherichia coli K12. J Gen Microbiol 132:1763–1766
    [Google Scholar]
  42. Wilson T. J. G., Bertrand N., Tang J.-L., Feng J.-X., Pan M.-Q., Barber C. E., Dow J. M., Daniels M. J. 1998; The rpfA gene of Xanthomonas campestris pathovar campestris , which is involved in the regulation of pathogenicity factor production, encodes an aconitase. Mol Microbiol 28:961–970 [CrossRef]
    [Google Scholar]
  43. Woodland M. P., Dalton H. 1984; Purification and characterization of component A of the methane monooxygenase from Methylococcus capsulatus (Bath). J Biol Chem 259:53–59
    [Google Scholar]
  44. Woods S. A., Miles J. S., Roberts R. E., Guest J. R. 1986; Structural and functional relationships between fumarase and aspartase: nucleotide sequences of the fumarase ( fumC ) and aspartase ( aspA ) genes of Escherichia coli K12. Biochem J 237:547–557
    [Google Scholar]
  45. Yu D., Ellis H. M., Lee E.-C., Jenkins N. A., Copeland N. G., Court D. L. 2000; An efficient recombination system for chromosome engineering in Escherichia coli. . Proc Natl Acad Sci USA 97:5978–5983 [CrossRef]
    [Google Scholar]
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