1887

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.

Loading

Article metrics loading...

/content/journal/micro/10.1099/00221287-148-1-133
2002-01-01
2019-10-20
Loading full text...

Full text loading...

/deliver/fulltext/micro/148/1/1480133a.html?itemId=/content/journal/micro/10.1099/00221287-148-1-133&mimeType=html&fmt=ahah

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., Plunkett, G., III, Bloch, C. A. & 14 other authors ( 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 (λfrdA) 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.
  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., Müller, U., Linder, M. I., Linder, D. & Buckel, W. ( 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]
http://instance.metastore.ingenta.com/content/journal/micro/10.1099/00221287-148-1-133
Loading
/content/journal/micro/10.1099/00221287-148-1-133
Loading

Data & Media loading...

This is a required field
Please enter a valid email address
Approval was a Success
Invalid data
An Error Occurred
Approval was partially successful, following selected items could not be processed due to error