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Epimerization and substrate gating by a TE domain in β-lactam antibiotic biosynthesis

Nature Chemical Biology volume 10, pages 251–258 (2014)Cite this article

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Abstract

Nonribosomal peptide synthetases are versatile engines of bioactive natural product biosynthesis that function according to the multiple carrier thiotemplate mechanism. C-terminal thioesterase (TE) domains of these giant modular proteins typically catalyze product release by hydrolysis or macrocyclization. We now report an unprecedented, dual-function TE that is involved in the biosynthesis of nocardicin A, which is the paradigm monocyclic β-lactam antibiotic. Contrary to our expectation, a stereodefined series of potential peptide substrates for the nocardicin TE domain failed to undergo hydrolysis. The stringent discrimination against peptide intermediates was overcome by prior monocyclic β-lactam formation at an L-seryl site. Kinetic data are interpreted such that the TE domain acts as a gatekeeper to hold the assembling peptide on an upstream domain until β-lactam formation takes place and then rapidly catalyzes epimerization, which has not been observed previously as a TE catalytic function, and thioesterase cleavage to discharge a fully fledged pentapeptide β-lactam harboring nocardicin G, the universal precursor of the nocardicins.

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Figure 1: Isopenicillin N and nocardicin A biosyntheses.
Figure 2: Substrates synthesized to probe the specificity of NocTE.
Figure 3: In vitro substrate profiling of NocTE.
Figure 4: 1H-NMR spectrometric analysis of NocTE with epi-nocardicin G and nocardicin G–SNAC.
Figure 5: Kinetic analysis and determination of NocTE as a dual epimerase and hydrolase.
Figure 6: Possible pathways to monocyclic β-lactam formation.

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References

  1. Aoki, H., Sakai, H., Kohsaka, M., Konomi, T. & Hosoda, J. Nocardicin A, a new monocyclic β-lactam antibiotic. I. Discovery, isolation and characterization. J. Antibiot. (Tokyo) 29, 492–500 (1976).

    Article  CAS  Google Scholar 

  2. Townsend, C.A. & Wilson, B.A. The role of nocardicin G in nocardicin A biosynthesis. J. Am. Chem. Soc. 110, 3320–3321 (1988).

    Article  CAS  Google Scholar 

  3. Townsend, C.A. & Brown, A.M. Nocardicin A: Biosynthetic experiments with amino acid precursors. J. Am. Chem. Soc. 105, 913–918 (1983).

    Article  CAS  Google Scholar 

  4. Townsend, C.A., Brown, A.M. & Nguyen, L.T. Nocardicin A: Stereochemical and biomimetic studies of monocyclic β-lactam formation. J. Am. Chem. Soc. 105, 919–927 (1983).

    Article  CAS  Google Scholar 

  5. Fawcett, P.A. et al. Synthesis of δ-(α-aminoadipyl)cysteinylvaline and its role in penicillin biosynthesis. Biochem. J. 157, 651–660 (1976).

    Article  CAS  Google Scholar 

  6. Samson, S.M. et al. Isolation, sequence determination and expression in Escherichia coli of the isopenicillin N synthetase gene from Cephalosporium acremonium. Nature 318, 191–194 (1985).

    Article  CAS  Google Scholar 

  7. Banko, G., Demain, A.L. & Wolfe, S. δ-(l-α-aminoadipyl)-L-cysteinyl-D-valine synthetase (ACV synthetase): a multifunctional enzyme with broad substrate specificity for the synthesis of penicillin and cephalosporin precursors. J. Am. Chem. Soc. 109, 2858–2860 (1987).

    Article  CAS  Google Scholar 

  8. Roach, P.L. et al. Structure of isopenicillin N synthase complexed with substrate and the mechanism of penicillin formation. Nature 387, 827–830 (1997).

    Article  CAS  Google Scholar 

  9. Townsend, C.A. & Brown, A.M. Nocardicin A biosynthesis: stereochemical course of monocyclic β-lactam formation. J. Am. Chem. Soc. 104, 1748–1750 (1982).

    Article  CAS  Google Scholar 

  10. Gunsior, M. et al. The biosynthetic gene cluster for a monocyclic β-lactam antibiotic, nocardicin A. Chem. Biol. 11, 927–938 (2004).

    Article  CAS  Google Scholar 

  11. Reeve, A.M., Breazeale, S.D. & Townsend, C.A. Purification, characterization, and cloning of an S-adenosylmethionine–dependent 3-amino-3-carboxypropyltransferase in nocardicin biosynthesis. J. Biol. Chem. 273, 30695–30703 (1998).

    Article  CAS  Google Scholar 

  12. Kelly, W.L. & Townsend, C.A. Role of the cytochrome P450 NocL in nocardicin A biosynthesis. J. Am. Chem. Soc. 124, 8186–8187 (2002).

    Article  CAS  Google Scholar 

  13. Kelly, W.L. Mutational analysis and characterization of nocardicin C-9′ epimerase. J. Biol. Chem. 279, 38220–38227 (2004).

    Article  CAS  Google Scholar 

  14. Kelly, W.L. & Townsend, C.A. Mutational analysis of nocK and nocL in the nocardicin A producer Nocardia uniformis. J. Bacteriol. 187, 739–746 (2005).

    Article  CAS  Google Scholar 

  15. Davidsen, J.M. & Townsend, C.A. Identification and characterization of NocR as a positive transcriptional regulator of the β-lactam nocardicin A in Nocardia uniformis. J. Bacteriol. 191, 1066–1077 (2009).

    Article  CAS  Google Scholar 

  16. Davidsen, J.M. & Townsend, C.A. In vivo characterization of nonribosomal peptide synthetases NocA and NocB in the biosynthesis of nocardicin A. Chem. Biol. 19, 297–306 (2012).

    Article  CAS  Google Scholar 

  17. Lambalot, R.H. et al. A new enzyme superfamily—the phosphopantetheinyl transferases. Chem. Biol. 3, 923–936 (1996).

    Article  CAS  Google Scholar 

  18. Stachelhaus, T., Hüser, A. & Marahiel, M.A. Biochemical characterization of peptidyl carrier protein (PCP), the thiolation domain of multifunctional peptide synthetases. Chem. Biol. 3, 913–921 (1996).

    Article  CAS  Google Scholar 

  19. Meier, J.L. & Burkart, M.D. The chemical biology of modular biosynthetic enzymes. Chem. Soc. Rev. 38, 2012–2045 (2009).

    Article  CAS  Google Scholar 

  20. Marahiel, M.A., Stachelhaus, T. & Mootz, H.D. Modular peptide synthetases involved in nonribosomal peptide synthesis. Chem. Rev. 97, 2651–2674 (1997).

    Article  CAS  Google Scholar 

  21. Walsh, C.T. et al. Tailoring enzymes that modify nonribosomal peptides during and after chain elongation on NRPS assembly lines. Curr. Opin. Chem. Biol. 5, 525–534 (2001).

    Article  CAS  Google Scholar 

  22. Sattely, E.S., Fischbach, M.A. & Walsh, C.T. Total biosynthesis: in vitro reconstitution of polyketide and nonribosomal peptide pathways. Nat. Prod. Rep. 25, 757 (2008).

    Article  CAS  Google Scholar 

  23. Du, L. & Lou, L. PKS and NRPS release mechanisms. Nat. Prod. Rep. 27, 255–278 (2010).

    Article  CAS  Google Scholar 

  24. Baltz, R.H. Function of MbtH homologs in nonribosomal peptide biosynthesis and applications in secondary metabolite discovery. J. Ind. Microbiol. Biotechnol. 38, 1747–1760 (2011).

    Article  CAS  Google Scholar 

  25. Davidsen, J.M., Bartley, D.M. & Townsend, C.A. Non-ribosomal propeptide precursor in nocardicin A biosynthesis predicted from adenylation domain specificity dependent on the MbtH family protein NocI. J. Am. Chem. Soc. 135, 1749–1759 (2013).

    Article  CAS  Google Scholar 

  26. Gaudelli, N.M. & Townsend, C.A. Stereocontrolled syntheses of peptide thioesters containing modified seryl residues as probes of antibiotic biosynthesis. J. Org. Chem. 78, 6412–6426 (2013).

    Article  CAS  Google Scholar 

  27. Trauger, J.W., Kohli, R.M., Mootz, H.D., Marahiel, M.A. & Walsh, C.T. Peptide cyclization catalysed by the thioesterase domain of tyrocidine synthetase. Nature 407, 215–218 (2000).

    Article  CAS  Google Scholar 

  28. Ehmann, D.E., Trauger, J.W., Stachelhaus, T. & Walsh, C.T. Aminoacyl-SNACs as small-molecule substrates for the condensation domains of nonribosomal peptide synthetases. Chem. Biol. 7, 765–772 (2000).

    Article  CAS  Google Scholar 

  29. Hamed, R.B. et al. The enzymes of β-lactam biosynthesis. Nat. Prod. Rep. 30, 21–107 (2013).

    Article  CAS  Google Scholar 

  30. Sieber, S.A., Walsh, C.T. & Marahiel, M.A. Loading peptidyl-coenzyme A onto peptidyl carrier proteins: a novel approach in characterizing macrocyclization by thioesterase domains. J. Am. Chem. Soc. 125, 10862–10866 (2003).

    Article  CAS  Google Scholar 

  31. Gokhale, R.S., Hunziker, D., Cane, D.E. & Khosla, C. Mechanism and specificity of the terminal thioesterase domain from the erythromycin polyketide synthase. Chem. Biol. 6, 117–125 (1999).

    Article  CAS  Google Scholar 

  32. Lu, H., Tsai, S.C., Khosla, C. & Cane, D.E. Expression, site-directed mutagenesis, and steady state kinetic analysis of the terminal thioesterase domain of the methymycin/picromycin polyketide synthase. Biochemistry 41, 12590–12597 (2002).

    Article  CAS  Google Scholar 

  33. Stachelhaus, T. & Walsh, C.T. Mutational analysis of the epimerization domain in the initiation module PheATE of gramicidin S synthetase. Biochemistry 39, 5775–5787 (2000).

    Article  CAS  Google Scholar 

  34. Linne, U. & Marahiel, M.A. Control of directionality in nonribosomal peptide synthesis: role of the condensation domain in preventing misinitiation and timing of epimerization. Biochemistry 39, 10439–10447 (2000).

    Article  CAS  Google Scholar 

  35. Amyes, T.L. & Richard, J.P. Generation and stability of a simple thiol ester enolate in aqueous solution. J. Am. Chem. Soc. 114, 10297–10302 (1992).

    Article  CAS  Google Scholar 

  36. Bordwell, F.G. Equilibrium acidities in dimethyl sulfoxide solution. Acc. Chem. Res. 21, 456–463 (1988).

    Article  CAS  Google Scholar 

  37. Balibar, C.J., Vaillancourt, F.H. & Walsh, C.T. Generation of D amino acid residues in assembly of arthrofactin by dual condensation/epimerization domains. Chem. Biol. 12, 1189–1200 (2005).

    Article  CAS  Google Scholar 

  38. Patel, H.M., Tao, J. & Walsh, C.T. Epimerization of an L-cysteinyl to a D-cysteinyl residue during thiazoline ring formation in siderophore chain elongation by pyochelin synthetase from Pseudomonas aeruginosa. Biochemistry 42, 10514–10527 (2003).

    Article  CAS  Google Scholar 

  39. Kopp, F. & Marahiel, M.A. Macrocyclization strategies in polyketide and nonribosomal peptide biosynthesis. Nat. Prod. Rep. 24, 735–749 (2007).

    Article  CAS  Google Scholar 

  40. Giraldes, J.W. et al. Structural and mechanistic insights into polyketide macrolactonization from polyketide-based affinity labels. Nat. Chem. Biol. 2, 531–536 (2006).

    Article  CAS  Google Scholar 

  41. Smith, S. The animal fatty acid synthase: one gene, one polypeptide, seven enzymes. FASEB J. 8, 1248–1259 (1994).

    Article  CAS  Google Scholar 

  42. Tsai, S.C. et al. Crystal structure of the macrocycle-forming thioesterase domain of the erythromycin polyketide synthase: versatility from a unique substrate channel. Proc. Natl. Acad. Sci. USA 98, 14808–14813 (2001).

    Article  CAS  Google Scholar 

  43. Korman, T.P. et al. Structure and function of an iterative polyketide synthase thioesterase domain catalyzing Claisen cyclization in aflatoxin biosynthesis. Proc. Natl. Acad. Sci. USA 107, 6246–6251 (2010).

    Article  CAS  Google Scholar 

  44. Newman, A.G., Vagstad, A.L., Belecki, K., Scheerer, J.R. & Townsend, C.A. Analysis of the cercosporin polyketide synthase CTB1 reveals a new fungal thioesterase function. Chem. Commun. (Camb.) 48, 11772–11774 (2012).

    Article  CAS  Google Scholar 

  45. Vagstad, A.L., Hill, E.A., Labonte, J.W. & Townsend, C.A. Characterization of a fungal thioesterase having Claisen cyclase and deacetylase activities in melanin biosynthesis. Chem. Biol. 19, 1525–1534 (2012).

    Article  CAS  Google Scholar 

  46. Vaillancourt, F.H., Yeh, E., Vosburg, D.A., O'Connor, S.E. & Walsh, C.T. Cryptic chlorination by a non-haem iron enzyme during cyclopropyl amino acid biosynthesis. Nature 436, 1191–1194 (2005).

    Article  CAS  Google Scholar 

  47. Salituro, G.M. & Townsend, C.A. Total syntheses of (−)-nocardicins A–G: a biogenetic approach. J. Am. Chem. Soc. 112, 760–770 (1990).

    Article  CAS  Google Scholar 

  48. Ho, S.N., Hunt, H.D., Horton, R.M., Pullen, J.K. & Pease, L.R. Site-directed mutagenesis by overlap extension using the polymerase chain reaction. Gene 77, 51–59 (1989).

    Article  CAS  Google Scholar 

  49. Collier, H.B. Letter: a note on the molar absorptivity of reduced Ellman's reagent, 3-carboxylato-4-nitrothiophenolate. Anal. Biochem. 56, 310–311 (1973).

    Article  CAS  Google Scholar 

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Acknowledgements

This work was supported by US National Institutes of Health grant AI014937. We thank K.A. Moshos, C.J. Hastings and J.W. Li for their helpful discussion, chemical advice and encouragement and R.F. Li for guidance with molecular biology. We also thank K. Belecki and I.P. Mortimer for high-resolution MS data and C.T. Walsh (Harvard University) for providing the pET29-Sfp expression plasmid. A. Majumdar is graciously acknowledged for his assistance with the 1H-NMR arrayed D2O-exchange experiment.

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  1. Department of Chemistry, Johns Hopkins University, Baltimore, Maryland, USA

    Nicole M Gaudelli & Craig A Townsend

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C.A.T. and N.M.G. developed the hypothesis and designed the study. N.M.G. performed all of the syntheses and experiments reported. Both authors analyzed and discussed the results. N.M.G. and C.A.T. prepared the manuscript.

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Correspondence to Craig A Townsend.

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Gaudelli, N., Townsend, C. Epimerization and substrate gating by a TE domain in β-lactam antibiotic biosynthesis. Nat Chem Biol 10, 251–258 (2014). https://doi.org/10.1038/nchembio.1456

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