Abstract
The streptothricin (ST) antibiotics, produced by Streptomyces bacteria, contain L-β-lysine ((3S)-3,6-diaminohexanoic acid) oligopeptides as pendant chains. Here we describe three unusual nonribosomal peptide synthetases (NRPSs) involved in ST biosynthesis: ORF 5 (a stand-alone adenylation (A) domain), ORF 18 (containing thiolation (T) and condensation (C) domains) and ORF 19 (a stand-alone A domain). We demonstrate that ST biosynthesis begins with adenylation of L-β-lysine by ORF 5, followed by transfer to the T domain of ORF 18. In contrast, L-β-lysine molecules adenylated by ORF 19 are used to elongate an L-β-lysine peptide chain on ORF 18, a reaction unexpectedly catalyzed by ORF 19 itself. Finally, the C domain of ORF 18 catalyzes the condensation of L-β-lysine oligopeptides covalently bound to ORF 18 with a freely diffusible intermediate to release the ST products. These results highlight an unusual activity for an A domain and unique mechanisms of crosstalk within NRPS machinery.
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Acknowledgements
We thank N. Iwasaki and J. Seta (Bruker Daltonics K.K.) for assistance with HPLC/Q-TOF MS data collection. Plasmids pKD46 and pCP 20 and E. coli BW25113 were supplied by H. Mori (Nara Institute of Science and Technology). This work was supported, in part, by Grant-in-Aid for Young Scientists (A) (22688007) from the Japan Society for the Promotion of Science (JSPS; to Y.H.), the Industrial Technology Research Grant Program in 2008 from the New Energy and Industrial Technology Development Organization of Japan (to Y.H.) and by Grant-in-Aid for Scientific Research on Innovative Area (23108520) from the JSPS (to Y.H.).
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C.M., T.U. and Y.H. conceived and designed the experiments; C.M., J.T, Y.K., M.I., M.T., K.S. and H.K. performed the experiments; and C.M. and Y.H. wrote the paper.
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Maruyama, C., Toyoda, J., Kato, Y. et al. A stand-alone adenylation domain forms amide bonds in streptothricin biosynthesis. Nat Chem Biol 8, 791–797 (2012). https://doi.org/10.1038/nchembio.1040
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DOI: https://doi.org/10.1038/nchembio.1040
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