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Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide


Phosphinothricin tripeptide (PTT, phosphinothricylalanylalanine) is a natural-product antibiotic and potent herbicide that is produced by Streptomyces hygroscopicus ATCC 21705 (ref. 1) and Streptomyces viridochromogenes DSM 40736 (ref. 2). PTT has attracted widespread interest because of its commercial applications and unique phosphinic acid functional group. Despite intensive study since its discovery in 1972 (see ref. 3 for a comprehensive review), a number of steps early in the PTT biosynthetic pathway remain uncharacterized. Here we report a series of interdisciplinary experiments involving the construction of defined S. viridochromogenes mutants, chemical characterization of accumulated intermediates, and in vitro assay of selected enzymes to examine these critical steps in PTT biosynthesis. Our results indicate that early PTT biosynthesis involves a series of catalytic steps that to our knowledge has not been described so far, including a highly unusual reaction for carbon bond cleavage. In sum, we define a pathway for early PTT biosynthesis that is more complex than previously appreciated.

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Figure 1: Map of PTT biosynthetic genes and model for early steps in PTT biosynthesis.
Figure 2: In vitro reconstitution of early PTT biosynthetic reactions monitored by 31P NMR.
Figure 3: An alternative pathway for CPEP biosynthesis from phosphonoacetaldehyde supported by data presented in this work.

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  1. Seto, H. et al. Studies on the biosynthesis of bialaphos (SF-1293). 1. Incorporation of 13C- and 2H-labeled precursors into bialaphos. J. Antibiot. (Tokyo) 35, 1719–1721 (1982).

    Article  CAS  Google Scholar 

  2. Bayer, E. et al. Phosphinothricin und phosphinothricyl-alanyl-alanin. Helv. Chim. Acta 55, 224–239 (1972).

    Article  CAS  Google Scholar 

  3. Thompson, C.J. & Seto, H. in Genetics and Biochemistry of Antibiotic Production (eds. Vining, L.C. & Stuttard, C.) 197–222 (Butterworth-Heinemann, Newton, Massachusetts, USA, 1995).

    Book  Google Scholar 

  4. Abell, L.M. & Villafranca, J.J. Investigation of the mechanism of phosphinothricin inactivation of Escherichia coli glutamine synthetase using rapid quench kinetic technique. Biochemistry 30, 6135–6141 (1991).

    Article  CAS  Google Scholar 

  5. Bailey, R.R. Brief overview of single-dose therapy for uncomplicated urinary tract infections. Chemotherapy 36 (suppl. 1), 27–30 (1990).

    Article  Google Scholar 

  6. Missinou, M.A. et al. Fosmidomycin for malaria. Lancet 360, 1941–1942 (2002).

    Article  CAS  Google Scholar 

  7. Blodgett, J.A., Zhang, J.K. & Metcalf, W.W. Molecular cloning, sequence analysis, and heterologous expression of the phosphinothricin tripeptide biosynthetic gene cluster from Streptomyces viridochromogenes DSM 40736. Antimicrob. Agents Chemother. 49, 230–240 (2005).

    Article  CAS  Google Scholar 

  8. Schwartz, D. et al. Biosynthetic gene cluster of the herbicide phosphinothricin tripeptide from Streptomyces viridochromogenes Tü494. Appl. Environ. Microbiol. 70, 7093–7102 (2004).

    Article  CAS  Google Scholar 

  9. Hidaka, T., Hidaka, M. & Seto, H. Studies on the biosynthesis of bialaphos (SF-1293). 14. Nucleotide sequence of phosphoenolpyruvate phosphomutase gene isolated from a bialaphos producing organism, Streptomyces hygroscopicus, and its expression in Streptomyces lividans. J. Antibiot. (Tokyo) 45, 1977–1980 (1992).

    Article  CAS  Google Scholar 

  10. Nakashita, H., Kozuka, K., Hidaka, T., Hara, O. & Seto, H. Identification and expression of the gene encoding phosphonopyruvate decarboxylase of Streptomyces hygroscopicus. Biochim. Biophys. Acta 1490, 159–162 (2000).

    Article  CAS  Google Scholar 

  11. Schwartz, D., Recktenwald, J., Pelzer, S. & Wohlleben, W. Isolation and characterization of the PEP-phosphomutase and the phosphonopyruvate decarboxylase genes from the phosphinothricin tripeptide producer Streptomyces viridochromogenes Tü494. FEMS Microbiol. Lett. 163, 149–157 (1998).

    CAS  PubMed  Google Scholar 

  12. Hidaka, T. et al. Studies on the biosynthesis of bialaphos (SF-1293). 11. Biochemical mechanism of C-P bond formation of bialaphos: use of gene manipulation for the analysis of the C-P bond formation step. Agric. Biol. Chem. 54, 2121–2125 (1990).

    CAS  PubMed  Google Scholar 

  13. Lee, S.H., Hidaka, T., Nakashita, H. & Seto, H. The carboxyphosphonoenolpyruvate synthase-encoding gene from the bialaphos-producing organism Streptomyces hygroscopicus. Gene 153, 143–144 (1995).

    Article  CAS  Google Scholar 

  14. Babbitt, P.C. et al. The enolase superfamily: a general strategy for enzyme-catalyzed abstraction of the α-protons of carboxylic acids. Biochemistry 35, 16489–16501 (1996).

    Article  CAS  Google Scholar 

  15. Hara, O. et al. The bialaphos biosynthetic genes of Streptomyces viridochromogenes: cloning, heterospecific expression, and comparison with the genes of Streptomyces hygroscopicus. J. Gen. Microbiol. 137, 351–359 (1991).

    Article  CAS  Google Scholar 

  16. Seto, H. et al. Studies on the biosynthesis of bialaphos (SF-1293). Part 3. Production of phosphinic acid derivatives, MP-103, MP-104 and MP-105, by a blocked mutant of Streptomyces hygroscopicus SF-1293 and their roles in the biosynthesis of bialaphos. Biochem. Biophys. Res. Commun. 111, 1008–1014 (1983).

    Article  CAS  Google Scholar 

  17. Alijah, R., Dorendorf, J., Talay, S., Pühler, A. & Wohlleben, W. Genetic analysis of the phosphinothricin-tripeptide biosynthetic pathway of Streptomyces viridochromogenes Tü494. Appl. Microbiol. Biotechnol. 34, 749–755 (1991).

    Article  CAS  Google Scholar 

  18. Imai, S., Seto, H., Ogawa, H., Satoh, A. & Otake, N. Studies on the biosynthesis of fosfomycin. Conversion of 2-hydroxyethylphosphonic acid and 2-aminoethylphosphonic acid to fosfomycin. Agric. Biol. Chem. 49, 873–874 (1985).

    CAS  Google Scholar 

  19. Taylor, P.P., Pantaleone, D.P., Senkpeil, R.F. & Fotheringham, I.G. Novel biosynthetic approaches to the production of unnatural amino acids using transaminases. Trends Biotechnol. 16, 412–418 (1998).

    Article  CAS  Google Scholar 

  20. Imai, S. et al. Studies on the biosynthesis of bialaphos (SF-1293). 4. Production of phosphonic acid derivatives, 2-hydroxyethylphosphonic acid, hydroxymethylphosphonic acid and phosphonoformic acid by blocked mutants of Streptomyces hygroscopicus SF-1293 and their roles in the biosynthesis of bialaphos. J. Antibiot. (Tokyo) 37, 1505–1508 (1984).

    Article  CAS  Google Scholar 

  21. Potters, M.B. et al. Phosphoprotein with phosphoglycerate mutase activity from the archaeon Sulfolobus solfataricus. J. Bacteriol. 185, 2112–2121 (2003).

    Article  CAS  Google Scholar 

  22. Seto, H. et al. Studies on the biosynthesis of fosfomycin. 2. Conversion of 2-hydroxypropylphosphonic acid to fosfomycin by blocked mutants of Streptomyces wedmorensis. J. Antibiot. (Tokyo) 44, 1286–1288 (1991).

    Article  CAS  Google Scholar 

  23. Woodyer, R.D., Li, G.Y., Zhao, H.M. & van der Donk, W.A. New insight into the mechanism of methyl transfer during the biosynthesis of fosfomycin. Chem. Commun. (Camb.) 359–361 (2007).

  24. Woodyer, R.D. et al. Heterologous production of fosfomycin and identification of the minimal biosynthetic gene cluster. Chem. Biol. 13, 1171–1182 (2006).

    Article  CAS  Google Scholar 

  25. Wanner, B.L. in Methods in Molecular Genetics Vol. 3 (ed. Adolph, K.W.) 291–310 (Academic, Orlando, Florida, USA, 1994).

    Google Scholar 

  26. Martinez, A. et al. Genetically modified bacterial strains and novel bacterial artificial chromosome shuttle vectors for constructing environmental libraries and detecting heterologous natural products in mutiple expression hosts. Appl. Environ. Microbiol. 70, 2452–2463 (2004).

    Article  CAS  Google Scholar 

  27. Keiser, T., Bibb, M.J., Buttner, M.J., Chater, K.F. & Hopwood, D.A. Practical Streptomyces Genetics (John Innes Centre, Norwich, England, 2000).

    Google Scholar 

  28. Hirsch, C.F. & Ensign, J.C. Heat activation of Streptomyces viridochromogenes spores. J. Bacteriol. 126, 24–30 (1976).

    CAS  PubMed  PubMed Central  Google Scholar 

  29. Vidal, G., Thiaudiere, E., Canioni, P. & Gallis, J.L. Aminomethylphosphonate and 2-aminoethylphosphonate as 31P-NMR pH markers for extracellular and cytosolic spaces in the isolated perfused rat liver. NMR Biomed. 13, 289–296 (2000).

    Article  CAS  Google Scholar 

  30. Vasavada, K.V., Ray, B.D. & Nageswara Rao, B.D. 31P NMR lineshapes of β-P (ATP) in the presence of Mg2+ and Ca2+: estimate of exchange rates. J. Inorg. Biochem. 21, 323–335 (1984).

    Article  CAS  Google Scholar 

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This work was supported by US National Institute of General Medical Sciences grants GM59334 and GM067725 and US National Institute of Health Chemical Biology Interface Training Grant 5T32 GM070421. The authors wish to thank A. Salyers (University of Illinois, Urbana-Champaign) for the gift of Bacteroides fragilis NCTC9343 genomic DNA and M.J. Thomas (University of Wisconsin, Madison) for plasmids pOJ260 and pKC1139. We also thank V. Mainz and P. Molitor for NMR advice and C. Wenger for developing custom data analysis software for MS (University of Illinois, Urbana-Champaign).

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W.W.M. and J.A.V.B. designed most experiments and wrote the manuscript. J.A.V.B. conducted all microbiological, genetic and molecular biological experiments. NMR analyses were conducted by J.A.V.B. and G.L. G.L., J.E.V. and W.A.V. designed and carried out chemical syntheses and analyses. P.M.T. and N.L.K. designed and conducted the mass spectrometry experiments.

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Correspondence to William W Metcalf.

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Supplementary Figures 1–10, Supplementary Tables 1 and 2, Supplementary Methods, Supplementary Data (PDF 703 kb)

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Blodgett, J., Thomas, P., Li, G. et al. Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide. Nat Chem Biol 3, 480–485 (2007).

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