Unusual transformations in the biosynthesis of the antibiotic phosphinothricin tripeptide

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Abstract

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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Acknowledgements

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).

Author information

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.

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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