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
2020-04-05
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 USA96:10412–10417[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 Biochem131:373–378[CrossRef]
    [Google Scholar]
  3. Beinert H., Kennedy M. C., Stout C. D.. 1996; Aconitase as iron–sulfur protein, enzyme and iron-regulatory protein. Chem Rev96:2335–2373[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 Bacteriol171:3494–3503
    [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 Biochem233:317–326[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. Science277:1453–1470[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 Microbiol35:961–973[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 Biochem268:3577–3586[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 Bacteriol134:1141–1156
    [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 Genet178:409–418[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 . Microbiology143:3795–3805[CrossRef]
    [Google Scholar]
  13. Flint D. H., Emptage M. H.. 1988; Dihydroxy acid dehydratase from spinach contains a [2Fe–2S] cluster. J Biol Chem263:3558–3564
    [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. Biochemistry31:10331–10337[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. Microbiology144:929–935[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 J316:887–892
    [Google Scholar]
  17. Gruer M. J., Guest J. R.. 1994; Two genetically-distinct and differentially-regulated aconitases (AcnA and AcnB) in Escherichia coli . Microbiology140:2531–2541[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 Sci22:3–6[CrossRef]
    [Google Scholar]
  19. Gruer M. J., Bradbury A. J., Guest J. R.. 1997b; Construction and properties of aconitase mutants of Escherichia coli . Microbiology143:1837–1846[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 USA93:8175–8182[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 Bacteriol179:928–940
    [Google Scholar]
  22. Horswill A. R., Escalante-Semerena J. C.. 1999a; Salmonella typhimurium LT2 catabolizes propionate via the 2-methylcitric acid cycle. J Bacteriol181:5615–5623
    [Google Scholar]
  23. Horswill A. R., Escalante-Semerena J. C.. 1999b; The prpE gene of Salmonella typhimurium LT2 encodes propionyl-CoA synthetase. Microbiology145:1381–1388[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. Biochemistry40:4703–4713[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 J344:739–746[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 Chem258:11098–11105
    [Google Scholar]
  27. Laemmli U. K.. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature277:680–685
    [Google Scholar]
  28. Lakshmi T. M., Helling R. B.. 1976; Selection for citrate synthase deficiency in icd mutants of Escherichia coli . J Bacteriol127:76–83
    [Google Scholar]
  29. Lennox E. S.. 1955; Transduction of linked genetic characters of host by bacteriophage P1. Virology1:190–206[CrossRef]
    [Google Scholar]
  30. Lauble H., Kennedy M. C., Beinert H., Stout C. D.. 1992; Crystal structures of aconitases with isocitrate and nitroisocitrate bound. Biochemistry31:2735–2748[CrossRef]
    [Google Scholar]
  31. Marsh P.. 1986; ptac-85, an Escherichia coli vector for expression of non-fusion proteins. Nucleic Acids Res14:3603[CrossRef]
    [Google Scholar]
  32. Miles J. M., Guest J. R.. 1984; Complete nucleotide sequence of the fumarase gene fumA of Escherichia coli . Nucleic Acids Res12:3631–3642[CrossRef]
    [Google Scholar]
  33. Prodromou C., Artymiuk P. J., Guest J. R.. 1992; The aconitase of Escherichia coli . Eur J Biochem204:599–609[CrossRef]
    [Google Scholar]
  34. Robbins A. H., Stout C. D.. 1989; The structure of aconitase. Proteins5:289–312[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 Biol80:297–314[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 Bacteriol181:1072–1078
    [Google Scholar]
  38. Tang Y., Guest J. R.. 1999; Direct evidence for mRNA binding and post-transcriptional regulation by Escherichia coli aconitases. Microbiology145:3069–3079
    [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 Microbiol168:428–436[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 Dis17:738–747[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 Microbiol132:1763–1766
    [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 Microbiol28:961–970[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 Chem259:53–59
    [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 J237:547–557
    [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 USA97:5978–5983[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...

Most cited this month

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