Abstract
Nonribosomal peptide synthetases (NRPSs) are multidomain modular biosynthetic assembly lines that polymerize amino acids into a myriad of biologically active nonribosomal peptides (NRPs). NRPS thioesterase (TE) domains employ diverse release strategies for off-loading thioester-tethered polymeric peptides from termination modules typically via hydrolysis, aminolysis, or cyclization to provide mature antibiotics as carboxylic acids/esters, amides, and lactams/lactones, respectively. Here we report the enzyme-catalyzed formation of a highly strained β-lactone ring during TE-mediated cyclization of a β-hydroxythioester to release the antibiotic obafluorin (Obi) from an NRPS assembly line. The Obi NRPS (ObiF) contains a type I TE domain with a rare catalytic cysteine residue that plays a direct role in β-lactone ring formation. We present a detailed genetic and biochemical characterization of the entire Obi biosynthetic gene cluster in plant-associated Pseudomonas fluorescens ATCC 39502 that establishes a general strategy for β-lactone biogenesis.
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Acknowledgements
This work was supported with start-up funds provided by Washington University in St. Louis and the Research Corporation for Science Advancement through a Cottrell Scholar award to T.A.W. We thank C.T. Walsh (Stanford ChEM-H) for productive scientific discussions. We thank Cofactor Genomics (St. Louis, MO) for bioinformatics consultation and genome sequence analysis. We thank S. Alvarez and B. Evans at the Proteomics & Mass Spectrometry Facility at the Donald Danforth Plant Science Center (St. Louis, MO) for the acquisition of high-resolution MS spectra (NSF Grant No. DBI-0922879). We thank J.-S. Taylor (WUSTL Department of Chemistry) for assistance with the [32P]PPi exchange assay. We thank J. Kao (WUSTL Department of Chemistry) for assistance with NMR experiments.
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T.A.W., J.E.S., and M.R.R. wrote the paper and prepared the supplementary information. T.A.W. oversaw all of the experiments. J.E.S. and M.R.R. cloned and purified ObiG, ObiH, and ObiL. J.E.S. functionally characterized ObiG, ObiH, and ObiL. M.R.R. cloned, purified, and functionally characterized ObiD and ObiF. J.E.S. and M.R.R. purified and characterized all compounds. T.A.W. and N.K.P. isolated Pseudomonas fluorescens gDNA, analyzed sequencing data, and annotated the Obi biosynthetic gene cluster. N.K.P. performed protein homology modeling and helped with preparation of the supplementary information.
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Schaffer, J., Reck, M., Prasad, N. et al. β-Lactone formation during product release from a nonribosomal peptide synthetase. Nat Chem Biol 13, 737–744 (2017). https://doi.org/10.1038/nchembio.2374
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DOI: https://doi.org/10.1038/nchembio.2374
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