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. 1995 Oct;177(19):5644–5652. doi: 10.1128/jb.177.19.5644-5652.1995

Purification, characterization, and genetic analysis of Mycobacterium tuberculosis urease, a potentially critical determinant of host-pathogen interaction.

D L Clemens 1, B Y Lee 1, M A Horwitz 1
PMCID: PMC177376  PMID: 7559354

Abstract

Mycobacterium tuberculosis urease (urea amidohydrolase [EC 3.5.1.5]) was purified and shown to contain three subunits: two small subunits, each approximately 11,000 Da, and a large subunit of 62,000 Da. The N-terminal sequences of the three subunits were homologous to those of the A, B, and C subunits, respectively, of other bacterial ureases. M. tuberculosis urease was specific for urea, with a Km of 0.3 mM, and did not hydrolyze thiourea, hydroxyurea, arginine, or asparagine. The enzyme was active over a broad pH range (optimal activity at pH 7.2) and was remarkably stable against heating to 60 degrees C and resistant to denaturation with urea. The enzyme was not inhibited by 1 mM EDTA but was inhibited by N-ethylmaleimide, hydroxyurea, acetohydroxamate, and phenylphosphorodiamidate. Urease activity was readily detectable in M. tuberculosis growing in nitrogen-rich broth, but expression increased 10-fold upon nitrogen deprivation, which is consistent with a role for the enzyme in nitrogen acquisition by the bacterium. The gene cluster encoding urease was shown to have organizational similarities to urease gene clusters of other bacteria. The nucleotide sequence of the M. tuberculosis urease gene cluster revealed open reading frames corresponding to the urease A, B, and C subunits, as well as to the urease accessory molecules F and G.

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Selected References

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  1. Armstrong J. A., Hart P. D. Response of cultured macrophages to Mycobacterium tuberculosis, with observations on fusion of lysosomes with phagosomes. J Exp Med. 1971 Sep 1;134(3 Pt 1):713–740. doi: 10.1084/jem.134.3.713. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. BRAUDE A. I., SIEMIENSKI J. Role of bacterial urease in experimental pyelonephritis. J Bacteriol. 1960 Aug;80:171–179. doi: 10.1128/jb.80.2.171-179.1960. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Breitenbach J. M., Hausinger R. P. Proteus mirabilis urease. Partial purification and inhibition by boric acid and boronic acids. Biochem J. 1988 Mar 15;250(3):917–920. doi: 10.1042/bj2500917. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Clemens D. L., Horwitz M. A. Characterization of the Mycobacterium tuberculosis phagosome and evidence that phagosomal maturation is inhibited. J Exp Med. 1995 Jan 1;181(1):257–270. doi: 10.1084/jem.181.1.257. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Crowle A. J., Dahl R., Ross E., May M. H. Evidence that vesicles containing living, virulent Mycobacterium tuberculosis or Mycobacterium avium in cultured human macrophages are not acidic. Infect Immun. 1991 May;59(5):1823–1831. doi: 10.1128/iai.59.5.1823-1831.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Gordon A. H., Hart P. D., Young M. R. Ammonia inhibits phagosome-lysosome fusion in macrophages. Nature. 1980 Jul 3;286(5768):79–80. doi: 10.1038/286079a0. [DOI] [PubMed] [Google Scholar]
  7. Griffith D. P., Musher D. M. Prevention of infected urinary stones by urease inhibition. Invest Urol. 1973 Nov;11(3):228–233. [PubMed] [Google Scholar]
  8. Hart P. D., Young M. R. Ammonium chloride, an inhibitor of phagosome-lysosome fusion in macrophages, concurrently induces phagosome-endosome fusion, and opens a novel pathway: studies of a pathogenic mycobacterium and a nonpathogenic yeast. J Exp Med. 1991 Oct 1;174(4):881–889. doi: 10.1084/jem.174.4.881. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Harth G., Clemens D. L., Horwitz M. A. Glutamine synthetase of Mycobacterium tuberculosis: extracellular release and characterization of its enzymatic activity. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9342–9346. doi: 10.1073/pnas.91.20.9342. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Jabri E., Carr M. B., Hausinger R. P., Karplus P. A. The crystal structure of urease from Klebsiella aerogenes. Science. 1995 May 19;268(5213):998–1004. [PubMed] [Google Scholar]
  11. Jones B. D., Mobley H. L. Proteus mirabilis urease: nucleotide sequence determination and comparison with jack bean urease. J Bacteriol. 1989 Dec;171(12):6414–6422. doi: 10.1128/jb.171.12.6414-6422.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Kobashi K., Takebe S., Numata A. Specific inhibition of urease by N-acylphosphoric triamides. J Biochem. 1985 Dec;98(6):1681–1688. doi: 10.1093/oxfordjournals.jbchem.a135439. [DOI] [PubMed] [Google Scholar]
  13. LARSON A. D., KALLIO R. E. Purification and properties of bacterial urease. J Bacteriol. 1954 Jul;68(1):67–73. doi: 10.1128/jb.68.1.67-73.1954. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Labigne A., Cussac V., Courcoux P. Shuttle cloning and nucleotide sequences of Helicobacter pylori genes responsible for urease activity. J Bacteriol. 1991 Mar;173(6):1920–1931. doi: 10.1128/jb.173.6.1920-1931.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. 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]
  16. Lee M. H., Mulrooney S. B., Renner M. J., Markowicz Y., Hausinger R. P. Klebsiella aerogenes urease gene cluster: sequence of ureD and demonstration that four accessory genes (ureD, ureE, ureF, and ureG) are involved in nickel metallocenter biosynthesis. J Bacteriol. 1992 Jul;174(13):4324–4330. doi: 10.1128/jb.174.13.4324-4330.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Lee M. H., Pankratz H. S., Wang S., Scott R. A., Finnegan M. G., Johnson M. K., Ippolito J. A., Christianson D. W., Hausinger R. P. Purification and characterization of Klebsiella aerogenes UreE protein: a nickel-binding protein that functions in urease metallocenter assembly. Protein Sci. 1993 Jun;2(6):1042–1052. doi: 10.1002/pro.5560020617. [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Lee T. D., Vemuri S. MacProMass: a computer program to correlate mass spectral data to peptide and protein structures. Biomed Environ Mass Spectrom. 1990 Nov;19(11):639–645. doi: 10.1002/bms.1200191103. [DOI] [PubMed] [Google Scholar]
  19. Lévy-Frébault V. V., Portaels F. Proposed minimal standards for the genus Mycobacterium and for description of new slowly growing Mycobacterium species. Int J Syst Bacteriol. 1992 Apr;42(2):315–323. doi: 10.1099/00207713-42-2-315. [DOI] [PubMed] [Google Scholar]
  20. Maeda M., Hidaka M., Nakamura A., Masaki H., Uozumi T. Cloning, sequencing, and expression of thermophilic Bacillus sp. strain TB-90 urease gene complex in Escherichia coli. J Bacteriol. 1994 Jan;176(2):432–442. doi: 10.1128/jb.176.2.432-442.1994. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Mahadevan S., Sauer F. D., Erfle J. D. Purification and properties of urease from bovine rumen. Biochem J. 1977 Jun 1;163(3):495–501. doi: 10.1042/bj1630495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Millner O. E., Jr, Andersen J. A., Appler M. E., Benjamin C. E., Edwards J. G., Humphrey D. T., Shearer E. M. Flurofamide: a potent inhibitor of bacterial urease with potential clinical utility in the treatment of infection induced urinary stones. J Urol. 1982 Feb;127(2):346–350. doi: 10.1016/s0022-5347(17)53779-9. [DOI] [PubMed] [Google Scholar]
  23. Mobley H. L., Cortesia M. J., Rosenthal L. E., Jones B. D. Characterization of urease from Campylobacter pylori. J Clin Microbiol. 1988 May;26(5):831–836. doi: 10.1128/jcm.26.5.831-836.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Mobley H. L., Garner R. M., Bauerfeind P. Helicobacter pylori nickel-transport gene nixA: synthesis of catalytically active urease in Escherichia coli independent of growth conditions. Mol Microbiol. 1995 Apr;16(1):97–109. doi: 10.1111/j.1365-2958.1995.tb02395.x. [DOI] [PubMed] [Google Scholar]
  25. Mobley H. L., Hausinger R. P. Microbial ureases: significance, regulation, and molecular characterization. Microbiol Rev. 1989 Mar;53(1):85–108. doi: 10.1128/mr.53.1.85-108.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Mulrooney S. B., Hausinger R. P. Sequence of the Klebsiella aerogenes urease genes and evidence for accessory proteins facilitating nickel incorporation. J Bacteriol. 1990 Oct;172(10):5837–5843. doi: 10.1128/jb.172.10.5837-5843.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Mulrooney S. B., Lynch M. J., Mobley H. L., Hausinger R. P. Purification, characterization, and genetic organization of recombinant Providencia stuartii urease expressed by Escherichia coli. J Bacteriol. 1988 May;170(5):2202–2207. doi: 10.1128/jb.170.5.2202-2207.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Park I. S., Carr M. B., Hausinger R. P. In vitro activation of urease apoprotein and role of UreD as a chaperone required for nickel metallocenter assembly. Proc Natl Acad Sci U S A. 1994 Apr 12;91(8):3233–3237. doi: 10.1073/pnas.91.8.3233. [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Pianotti R. S., Mohan R. R., Schwartz B. S. Proteus vulgaris urease: in vitro inhibition by urea analogues. Proc Soc Exp Biol Med. 1966 Jun;122(2):506–508. doi: 10.3181/00379727-122-31174. [DOI] [PubMed] [Google Scholar]
  30. Saraste M., Sibbald P. R., Wittinghofer A. The P-loop--a common motif in ATP- and GTP-binding proteins. Trends Biochem Sci. 1990 Nov;15(11):430–434. doi: 10.1016/0968-0004(90)90281-f. [DOI] [PubMed] [Google Scholar]
  31. Schmitz A., Gartemann K. H., Fiedler J., Grund E., Eichenlaub R. Cloning and sequence analysis of genes for dehalogenation of 4-chlorobenzoate from Arthrobacter sp. strain SU. Appl Environ Microbiol. 1992 Dec;58(12):4068–4071. doi: 10.1128/aem.58.12.4068-4071.1992. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Schägger H., von Jagow G. Tricine-sodium dodecyl sulfate-polyacrylamide gel electrophoresis for the separation of proteins in the range from 1 to 100 kDa. Anal Biochem. 1987 Nov 1;166(2):368–379. doi: 10.1016/0003-2697(87)90587-2. [DOI] [PubMed] [Google Scholar]
  33. Smoot D. T., Mobley H. L., Chippendale G. R., Lewison J. F., Resau J. H. Helicobacter pylori urease activity is toxic to human gastric epithelial cells. Infect Immun. 1990 Jun;58(6):1992–1994. doi: 10.1128/iai.58.6.1992-1994.1990. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Todd M. J., Hausinger R. P. Competitive inhibitors of Klebsiella aerogenes urease. Mechanisms of interaction with the nickel active site. J Biol Chem. 1989 Sep 25;264(27):15835–15842. [PubMed] [Google Scholar]
  35. Todd M. J., Hausinger R. P. Purification and characterization of the nickel-containing multicomponent urease from Klebsiella aerogenes. J Biol Chem. 1987 May 5;262(13):5963–5967. [PubMed] [Google Scholar]
  36. Todd M. J., Hausinger R. P. Reactivity of the essential thiol of Klebsiella aerogenes urease. Effect of pH and ligands on thiol modification. J Biol Chem. 1991 Jun 5;266(16):10260–10267. [PubMed] [Google Scholar]
  37. Ullrich A., Berman C. H., Dull T. J., Gray A., Lee J. M. Isolation of the human insulin-like growth factor I gene using a single synthetic DNA probe. EMBO J. 1984 Feb;3(2):361–364. doi: 10.1002/j.1460-2075.1984.tb01812.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Xu S., Cooper A., Sturgill-Koszycki S., van Heyningen T., Chatterjee D., Orme I., Allen P., Russell D. G. Intracellular trafficking in Mycobacterium tuberculosis and Mycobacterium avium-infected macrophages. J Immunol. 1994 Sep 15;153(6):2568–2578. [PubMed] [Google Scholar]
  39. van Anken H. C., Schiphorst M. E. A kinetic determination of ammonia in plasma. Clin Chim Acta. 1974 Oct 30;56(2):151–157. doi: 10.1016/0009-8981(74)90223-x. [DOI] [PubMed] [Google Scholar]

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