1887

Abstract

This study describes the cloning, genetic analysis and biochemical characterization of a leucyl aminopeptidase (LAP) from . A gene encoding LAP was cloned from and the expressed 55 kDa protein displayed homology to aminopeptidases from Gram-negative bacteria, plants and mammals. This LAP demonstrated amidolytic activity against -leucine--nitroanilide. Optimal activity was observed at pH 8·0 and 45 °C, with of 232 μmol min (mg protein) and of 0·65 mM. The data suggest that LAP could be allosteric ( =2·27), with regulatory homohexamers, and its activity was inhibited by ion chelators and enhanced by divalent manganese, cobalt and nickel cations. Bestatin inhibited both LAP activity (IC=49·9 nM) and growth . The results point to the potential use of LAP as a drug target to develop novel anti- agents.

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2005-06-01
2019-10-15
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References

  1. Alm, R. A., Ling, L. S., Moir, D. T. & 20 other authors ( 1999; ). Genomic-sequence comparison of two unrelated isolates of the human gastric pathogen Helicobacter pylori. Nature 397, 176–180.[CrossRef]
    [Google Scholar]
  2. Björkholm, B., Sjölund, M., Falk, P. G., Berg, O. G., Engstrand, L. & Andersson, D. I. ( 2001; ). Mutation frequency and biological cost of antibiotic resistance in Helicobacter pylori. Proc Natl Acad Sci U S A 98, 14607–14612.[CrossRef]
    [Google Scholar]
  3. Booth, M., Jennings, V., Fhaolain, I. N. & O'Cuinn, G. ( 1990; ). Prolidase activity of Lactococcus lactis subsp. cremoris AM2: partial purification and characterization. J Dairy Res 57, 245–254.[CrossRef]
    [Google Scholar]
  4. Carpenter, F. H. & Vahl, J. M. ( 1973; ). Leucine aminopeptidase (bovine lens). Mechanism of activation by Mg2+ and Mn2+ of the zinc metalloenzyme, amino acid composition, and sulfhydryl content. J Biol Chem 248, 294–304.
    [Google Scholar]
  5. Cheng, N., Xie, J. S., Zhang, M. Y., Shu, C. & Zhu, D. X. ( 2003; ). A specific anti-Helicobacter pylori agent NE2001: synthesis and its effect on the growth of H. pylori. Bioorg Med Chem Lett 13, 2703–2707.[CrossRef]
    [Google Scholar]
  6. Cottrell, G. S., Hooper, N. M. & Turner, A. J. ( 2000; ). Cloning, expression, and characterization of human cytosolic aminopeptidase P: a single manganese (II)-dependent enzyme. Biochemistry 39, 15121–15128.[CrossRef]
    [Google Scholar]
  7. Cover, T. L. & Blaser, M. J. ( 1996; ). Helicobacter pylori infection, a paradigm for chronic mucosal inflammation: pathogenesis and implications for eradication and prevention. Adv Int Med 41, 85–117.
    [Google Scholar]
  8. Dixon, M. & Webb, E. C. ( 1979; ). Enzymes, 3rd edn. London: Longman.
  9. D'Souza, V. M. & Holz, R. C. ( 1999; ). The methionyl aminopeptidase from Escherichia coli can function as an iron (II) enzyme. Biochemistry 38, 11079–11085.[CrossRef]
    [Google Scholar]
  10. Ellis, K. J. & Morrison, J. F. ( 1982; ). Buffers of constant ionic strength for studying pH-dependent processes. Methods Enzymol 87, 405–426.
    [Google Scholar]
  11. Goldberg, A. L. & Dice, J. F. ( 1974; ). Intracellular protein degradation in mammalian and bacterial cells. Annu Rev Biochem 43, 835–869.[CrossRef]
    [Google Scholar]
  12. Goldberg, A. L. & John, A. C. S. ( 1976; ). Intracellular protein degradation in mammalian and bacterial cells: part 2. Annu Rev Biochem 45, 747–803.[CrossRef]
    [Google Scholar]
  13. Gu, Y. G. & Walling, L. L. ( 2002; ). Identification of residues critical for activity of the wound-induced leucine aminopeptidase (LAP-A) of tomato. Eur J Biochem 269, 1630–1640.[CrossRef]
    [Google Scholar]
  14. Hopkins, R. J., Girardi, L. S. & Turney, E. A. ( 1996; ). Relationship between Helicobacter pylori eradication and reduced duodenal and gastic ulcer recurrence: a review. Gastroenterology 110, 1244–1252.[CrossRef]
    [Google Scholar]
  15. Howarth, J. & Lloyd, D. G. ( 2000; ). Simple 1,2-aminoalcohols as strain-specific antimalarial agents. J Antimicrob Chemother 46, 625–627.[CrossRef]
    [Google Scholar]
  16. Kim, H. & Lipscomb, W. N. ( 1993; ). Differentiation and identification of the two catalytic metal binding sites in bovine lens leucine aminopeptidase by X-ray crystallography. Proc Natl Acad Sci U S A 90, 5006–5010.[CrossRef]
    [Google Scholar]
  17. Knowles, G. ( 1993; ). The effects of arphamenine-A, an inhibitor of aminopeptidases, on in-vitro growth of Trypanosoma brucei brucei. J Antimicrob Chemother 32, 172–174.[CrossRef]
    [Google Scholar]
  18. Kreiss, C., Blum, A. L. & Malfertheiner, P. ( 1995; ). Peptic ulcer pathogenesis. Curr Opin Gastroenterol 11, 25–31.[CrossRef]
    [Google Scholar]
  19. Lazdunski, A. ( 1989; ). Peptidase and protease of Escherichia coli and Salmonella typhimurium. FEMS Microbiol Rev 63, 265–276.
    [Google Scholar]
  20. Malfertheiner, P., Megraud, F., O'Morain, C., Hungin, A. P., Jones, R., Axon, A., Graham, D. Y. & Tytgat, G. ( 2002; ). Current concepts in the management of Helicobacter pylori infection – the Maastricht 2-2000 Consensus Report. Aliment Pharmacol Ther 16, 167–180.
    [Google Scholar]
  21. Miller, C. G. ( 1975; ). Peptidases and proteases of Escherichia coli and Salmonella typhimurium. Annu Rev Microbiol 29, 485–504.[CrossRef]
    [Google Scholar]
  22. Morty, R. E. & Morehead, J. ( 2002; ). Cloning and characterization of a leucyl aminopeptidase from three pathogenic Leishmania species. J Biol Chem 277, 26057–26065.[CrossRef]
    [Google Scholar]
  23. Müller, K. D., von Recklinghausen, G., Heintschel von Heinegg, E. & Ansorg, R. ( 1991; ). Flocculation of venereal disease research laboratory reagent by Helicobacter pylori. Eur J Microbiol Infect Dis 10, 768–770.[CrossRef]
    [Google Scholar]
  24. Nankya-Kitaka, M. F., Curley, G. P., Gavigan, C. S., Bell, A. & Dalton, J. P. ( 1998; ). Plasmodium chabaudi chabaudi and P. falciparum: inhibition of aminopeptidase and parasite growth by bestatin and nitrobestatin. Parasitol Res 84, 552–558.[CrossRef]
    [Google Scholar]
  25. Niven, G. W. ( 1991; ). Purification and characterization of aminopeptidase A from Lactococcus lactis subsp. lactis NCDO712. J Gen Microbiol 137, 1207–1212.[CrossRef]
    [Google Scholar]
  26. Rawlings, N. D. & Barrett, A. J. ( 1995; ). Evolutionary families of metallopeptidases. Methods Enzymol 248, 183–228.
    [Google Scholar]
  27. Rogers, A. H., Gunadi, A., Gully, N. J. & Zilm, P. S. ( 1998; ). An aminopeptidase nutritionally important to Fusobacterium nucleatum. Microbiology 144, 1807–1813.[CrossRef]
    [Google Scholar]
  28. Sakakibara, T., Ito, K., Irie, Y., Hagiwara, T., Sakai, Y., Hayashi, M., Kishi, H., Sakamoto, M. & Suzuki, M. ( 1983; ). Toxicological studies on bestatin. I. Acute toxicity test in mice, rats and dogs. Jpn J Antibiot 36, 2971–2984.
    [Google Scholar]
  29. Smid, E. J., Poolman, B. & Konings, W. N. ( 1991; ). Casein utilization by lactococci. Appl Environ Microbiol 57, 2447–2453.
    [Google Scholar]
  30. Tan, P. S. T. & Konings, W. N. ( 1990; ). Purification and characterization of an aminopeptidase from Lactococcus lactis subsp. cremoris Wg2. Appl Environ Microbiol 56, 526–532.
    [Google Scholar]
  31. Taylor, A. ( 1993; ). Aminopeptidases: structure and function. FASEB J 7, 290–298.
    [Google Scholar]
  32. Taylor, A., Peltier, C. Z., Torre, F. J. & Hakamian, N. ( 1993; ). Inhibition of bovine lens leucine aminopeptidases by bestatin: number of binding sites and slow binding of this inhibitor. Biochemistry 32, 784–790.[CrossRef]
    [Google Scholar]
  33. Thierry, G. & Janine, R. B. ( 1996; ). Bacterial aminopeptidases: properties and functions. FEMS Microbiol Rev 18, 319–344.[CrossRef]
    [Google Scholar]
  34. Tomb, J. F., White, O., Kerlavage, A. R. & 39 other authors ( 1997; ). The complete genome sequence of the gastric pathogen Helicobacter pylori. Nature 388, 539–547.[CrossRef]
    [Google Scholar]
  35. Van Wart, H. E. & Lin, S. H. ( 1981; ). Metal binding stoichiometry and mechanism of metal ion modulation of the activity of porcine kidney leucine aminopeptidase. Biochemistry 20, 5682–5689.[CrossRef]
    [Google Scholar]
  36. Walker, K. W. & Bradshaw, R. A. ( 1998; ). Yeast methionine aminopeptidase I can utilize either Zn2+ or Co2+ as a cofactor: a case of mistaken identity? Protein Sci 7, 2684–2687.[CrossRef]
    [Google Scholar]
  37. Wilkes, S. H. & Prescott, J. M. ( 1985; ). The slow, tight binding of bestatin and amastatin to aminopeptidases. J Biol Chem 260, 13154–13162.
    [Google Scholar]
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