1887

Abstract

It was demonstrated previously that the operon consisting of the non-ribosomal peptide synthetase (NRPS) gene coupled with the polyketide synthase (PKS) gene involved in cyclic heptapeptide microcystin synthesis includes two different D-amino acid synthetase genes, an epimerization domain at the 3′ end of module 2, and the racemase gene . To determine the role of in microcystin synthesis, gene-disruption and complementation analyses were carried out. Insertional mutagenesis in the gene, generated by homologous recombination, abolished only microcystin synthesis, but did not influence cell growth. Furthermore, McyF supported D-Glu-independent growth of a strain of defective in D-Glu synthesis. It is concluded that is the glutamic acid racemase gene involved in the synthesis of D-Glu residues in the microcystin molecule. This is the first report of the racemase in prokaryotic NRPS.

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2001-05-01
2020-09-26
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References

  1. Carmichael W. W. 1994; The toxins of cyanobacteria. Sci Am270:64–72[CrossRef]
    [Google Scholar]
  2. Cheng Y., Walton J. D. 2000; A eukaryotic alanine racemase gene involved in cyclic peptide biosynthesis. J Biol Chem275:4906–4911[CrossRef]
    [Google Scholar]
  3. Fotheringham I. G., Bledig S. A., Taylor P. P. 1998; Characterization of the genes encoding d-amino acid transaminase and glutamate racemase, two d-glutamate biosynthetic enzymes of Bacillus sphaericus ATCC 10208. J Bacteriol180:4319–4323
    [Google Scholar]
  4. Harada K., Ogawa K., Matsuura K.. 7 other authors 1991; Isolation of two toxic heptapeptide microcystins from an axenic strain of Microcystis aeruginosa K-139. Toxicon29:479–489[CrossRef]
    [Google Scholar]
  5. Hoffmann K., Scheider-Scherzer E., Kleinkauf H., Zocher R. 1994; Purification and characterization of eucaryotic alanine racemase acting as key enzyme in cyclosporin biosynthesis. J Biol Chem269:12710–12714
    [Google Scholar]
  6. Jochimsen E. M., Carmichael W. W., An J. S.. 9 other authors 1998; Liver failure and death after exposure to microcystins at a hemodialysis center in Brazil. N Engl J Med338:873–878[CrossRef]
    [Google Scholar]
  7. Laemmli U. K. 1970; Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature227:680–685[CrossRef]
    [Google Scholar]
  8. Liu L., Yoshimura T., Endo K., Esaki N., Soda K. 1997; Cloning and expression of the glutamate racemase gene of Bacillus pumilus. J Biochem121:1156–1161
    [Google Scholar]
  9. Lugtenberg E. J. J., Wijsman H. J. W., van Zaane D. 1973; Properties of a d-glutamic acid-requiring mutant of Escherichia coli. J Bacteriol114:499–506
    [Google Scholar]
  10. MacKintosh C., Beatte K. A., Klumpp S., Cohen P., Codd G. A. 1990; Cyanobacterial microcystin-LR is a potent and specific inhibitor of protein phosphatases 1 and 2A from both mammals and higher plants. FEBS Lett264:187–192[CrossRef]
    [Google Scholar]
  11. Malathi K. C., Wachi M., Nagai K. 1999; Isolation of the murI gene from Brevibacterium lactofermentum ATCC 13869 encoding d-glutamate racemase. FEMS Microbiol Lett175:193–196
    [Google Scholar]
  12. Marahiel M. A. 1997; Protein templates for the biosynthesis of peptide antibiotics. Chem Biol4:561–567[CrossRef]
    [Google Scholar]
  13. Moore R. E., Chen J. L., Moore B. S., Patterson G. M. L. 1991; Biosynthesis of Microcystin-LR. Origin of the carbons in the Adda and Masp units. J Am Chem Soc113:5083–5084[CrossRef]
    [Google Scholar]
  14. Nishizawa T., Asayasma M., Fujii K., Harada K.-I., Shirai M. 1999; Genetic analysis of the peptide synthetase genes for a cyclic heptapeptide microcystin in Microcystis spp. J Biochem126:520–529[CrossRef]
    [Google Scholar]
  15. Nishizawa T., Ueda A., Asayasma M., Fujii K., Harada K.-I., Ochi K., Shirai M. 2000; Polyketide synthase gene coupled to the peptide synthetase module involved in the biosynthesis of the cyclic heptapeptide microcystin. J Biochem127:779–789[CrossRef]
    [Google Scholar]
  16. Pucci M. J., Thanassi J. A., Ho H., Falk P. J., Dougherty T. J. 1995; Staphylococcus haemolyticus contains two d-glutamic acid biosynthetic activities, a glutamate racemase and a d-amino acid transaminase. J Bacteriol177:336–342
    [Google Scholar]
  17. Sakamoto T., Shirai M., Asayama M., Aida T., Sato A., Tanaka K., Takahashi H., Nakano M. 1993; Characteristics of DNA and multiple rpoD homologs of Microcystis ( Synechocystis ) strains. Int J Syst Bacteriol43:844–847[CrossRef]
    [Google Scholar]
  18. 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]
  19. Shirai M., Ohtake A., Sano T.. 8 other authors 1991; Toxicity and toxins of natural blooms and isolated Microcystis spp. (Cyanobacteria) and improved procedure for purification of cultures. Appl Environ Microbiol57:1241–1245
    [Google Scholar]
  20. Simon R. 1984; High frequency mobilization of gram-negative bacterial replicons by the in vitro constructed Tn5-Mob transposon. Mol Gen Genet196:413–420[CrossRef]
    [Google Scholar]
  21. Sivonen K., Jones G. 1999; Cyanobacterial toxins. In Toxic Cyanobacteria in Water: a Guide to their Public Health Consequences, Monitoring and Management pp41–111 Edited by Chorus I., Bertram J.. London: E. & F. N. Spon;
    [Google Scholar]
  22. Stachelhaus T., Marahiel M. A. 1995; Modular structure of genes encoding multifunctional peptide synthetases required for non-ribosomal peptide synthesis. FEMS Microbiol Lett125:3–14[CrossRef]
    [Google Scholar]
  23. Stein T., Kluge B., Vater J., Franke P., Otto A., Wittmann-Liebold B. 1995; Gramicidin S synthetase 1 (phenylalanine racemase), a prototype of amino acid racemases containing the cofactor 4′-phosphopantetheine. Biochemistry34:4633–4642[CrossRef]
    [Google Scholar]
  24. Takahashi I., Hayano D., Asayama M., Futagami M., Watahiki M., Shirai M. 1996; Restriction barrier composed of an extracellular nuclease and restriction endonuclease in the unicellular cyanobacterium Microcystis sp. FEMS Microbiol Lett145:107–111[CrossRef]
    [Google Scholar]
  25. Watanabe M. F., Harada K., Carmichael W. W., Fujiki H. (editors) 1996; Toxic Microcystis Boca Raton, FL: CRC Press;
    [Google Scholar]
  26. Yohda M., Endo I., Abe Y., Ohta T., Iida T., Maruyama T., Kagawa Y. 1996; Gene for aspartate racemase from the sulfur-dependent hyperthermophilic archaeum, Desulfurococcus strain SY. J Biol Chem271:22017–22021[CrossRef]
    [Google Scholar]
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