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β-Lactone formation during product release from a nonribosomal peptide synthetase

Nature Chemical Biology volume 13, pages 737–744 (2017)Cite this article

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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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Figure 1: Biosynthetic gene cluster for obafluorin β-lactone from P. fluorescens ATCC 39502.
Figure 2: Enzymatic conversion of PAPPA to β-OH-p-NO2-homoPhe using recombinant oxidase ObiL, decarboxylase ObiG, and aldolase ObiH.
Figure 3: Enzymatic conversion of β-OH-p-NO2-homoPhe and 2,3-DHB to Obi β-lactone using recombinant NRPS assembly line ObiF and ObiD.
Figure 4: ObiF catalysis is required for β-lactone ring formation.
Figure 5: Model for Obi biosynthesis and the catalytic cycle of NRPS assembly line ObiF.

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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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  1. Jason E Schaffer and Margaret R Reck: These authors contributed equally to this work.

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  1. Department of Chemistry, Washington University in St. Louis, St. Louis, Missouri, USA

    Jason E Schaffer, Margaret R Reck, Neha K Prasad & Timothy A Wencewicz

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Contributions

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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Correspondence to Timothy A Wencewicz.

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Supplementary Results, Supplementary Tables 1–13 and Supplementary Figures 1–23 (PDF 11727 kb)

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Characterization of Chemical Compounds (PDF 4074 kb)

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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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