Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme

Nature Chemical Biology volume 14, pages 730–737 (2018)Cite this article

Abstract

Acyl-coenzyme A (CoA) ligases catalyze the activation of carboxylic acids via a two-step reaction of adenylation followed by thioesterification. Here, we report the discovery of a non-adenylating acyl-CoA ligase PtmA2 and the functional separation of an acyl-CoA ligase reaction. Both PtmA1 and PtmA2, two acyl-CoA ligases from the biosynthetic pathway of platensimycin and platencin, are necessary for the two steps of CoA activation. Gene inactivation of ptmA1 and ptmA2 resulted in the accumulation of free acid and adenylate intermediates, respectively. Enzymatic and structural characterization of PtmA2 confirmed its ability to only catalyze thioesterification. Structural characterization of PtmA2 revealed it binds both free acid and adenylate substrates and undergoes the established mechanism of domain alternation. Finally, site-directed mutagenesis restored both the adenylation and complete CoA activation reactions. This study challenges the currently accepted paradigm of adenylating enzymes and inspires future investigations on functionally separated acyl-CoA ligases and their ramifications in biology.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Fig. 1: Acyl-CoA ligases catalyze adenylation and thioesterification.
Fig. 2: Structures of PTM, PTN, and congeners and metabolite profiles of selected S. platensis strains upon LC–MS analysis.
Fig. 3: Functional separation of CoA activation in PTM and PTN biosynthesis.
Fig. 4: Overall structure and active sites of PtmA2 in both the adenylation and thioesterification conformations.
Fig. 5: Reconstitution of the lost adenylation activity of PtmA2 via site-directed mutagenesis.

Similar content being viewed by others

References

  1. Houten, S. M. & Wanders, R. J. A. A general introduction to the biochemistry of mitochondrial fatty acid β-oxidation. J. Inherit. Metab. Dis. 33, 469–477 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  2. Umbarger, H. E. Amino acid biosynthesis and its regulation. Annu. Rev. Biochem. 47, 532–606 (1978).

    Article  CAS  PubMed  Google Scholar 

  3. Ibba, M. & Soll, D. Aminoacyl-tRNA synthesis. Annu. Rev. Biochem. 69, 617–650 (2000).

    Article  CAS  PubMed  Google Scholar 

  4. Weissman, K. J. The structural biology of biosynthetic megaenzymes. Nat. Chem. Biol. 11, 660–670 (2015).

    Article  CAS  PubMed  Google Scholar 

  5. Hur, G. H., Vickery, C. R. & Burkart, M. D. Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology. Nat. Prod. Rep. 29, 1074–1098 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Gulick, A. M. Conformational dynamics in the acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chem. Biol. 4, 811–827 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Schmelz, S. & Naismith, J. H. Adenylate-forming enzymes. Curr. Opin. Struct. Biol. 19, 666–671 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Estrada, P. et al. The pimeloyl-CoA synthetase BioW defines a new fold for adenylate-forming enzymes. Nat. Chem. Biol. 13, 668–674 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Wang, M. et al. Using the pimeloyl-CoA synthetase adenylation fold to synthesize fatty acid thioesters. Nat. Chem. Biol. 13, 660–667 (2017).

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  11. Wu, R., Reger, A. S., Lu, X., Gulick, A. M. & Dunaway-Mariano, D. The mechanism of domain alternation in the acyl-adenylate forming ligase superfamily member 4-chlorobenzoate: coenzyme A ligase. Biochemistry 48, 4115–4125 (2009).

    Article  CAS  PubMed  Google Scholar 

  12. Rudolf, J. D., Dong, L.-B. & Shen, B. Platensimycin and platencin: inspirations for chemistry, biology, enzymology, and medicine. Biochem. Pharmacol. 133, 139–151 (2017).

    Article  CAS  PubMed  Google Scholar 

  13. Dong, L.-B. et al. In vivo instability of platensimycin and platencin: synthesis and biological evaluation of urea- and carbamate-platensimycin. Bioorg. Med. Chem. 25, 1990–1996 (2017).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Rudolf, J. D., Dong, L.-B., Manoogian, K. & Shen, B. Biosynthetic origin of the ether ring in platensimycin. J. Am. Chem. Soc. 138, 16711–16721 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  15. Smanski, M. J. et al. Dedicated ent-kaurene and ent-atiserene synthases for platensimycin and platencin biosynthesis. Proc. Natl. Acad. Sci. USA 108, 13498–13503 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  16. Rudolf, J. D., Dong, L.-B., Huang, T. & Shen, B. A genetically amenable platensimycin- and platencin-overproducer as a platform for biosynthetic explorations: a showcase of PtmO4, a long-chain acyl-CoA dehydrogenase. Mol. Biosyst. 11, 2717–2726 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Smanski, M. J. et al. Expression of the platencin biosynthetic gene cluster in heterologous hosts yielding new platencin congeners. J. Nat. Prod. 75, 2158–2167 (2012).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  18. Hedden, P. & Sponsel, V. A century of gibberellin research. J. Plant Growth Regul. 34, 740–760 (2015).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    Article  CAS  PubMed  Google Scholar 

  20. Gust, B., Challis, G. L., Fowler, K., Kieser, T. & Chater, K. F. PCR-targeted Streptomyces gene replacement identifies a protein domain needed for biosynthesis of the sesquiterpene soil odor geosmin. Proc. Natl. Acad. Sci. USA 100, 1541–1546 (2003).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Kieser, T., Bibb, M. J., Buttner, M. J., Chater, K. F. & Hopwood, D. A. Practical Streptomyces Genetics (The John Innes Foundation, Norwich, U.K., 2000)..

  22. MacNeil, D. J. et al. Analysis of Streptomyces avermitilis genes required for avermectin biosynthesis utilizing a novel integration vector. Gene 111, 61–68 (1992).

    Article  CAS  PubMed  Google Scholar 

  23. Dong, L.-B., Rudolf, J. D. & Shen, B. Antibacterial sulfur-containing platensimycin and platencin congeners from Streptomyces platensis SB12029. Bioorg. Med. Chem. 24, 6348–6353 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Herz, W., Kulanthaivel, P. & Watanabe, K. ent-Kauranes and other constituents of three Helianthus species. Phytochemistry 22, 2021–2025 (1983).

    Article  CAS  Google Scholar 

  25. Hindra et al. Strain prioritization for natural product discovery by a high-throughput real-time PCR method. J. Nat. Prod. 77, 2296–2303 (2014).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  26. Krissinel, E. & Henrick, K. Inference of macromolecular assemblies from crystalline state. J. Mol. Biol. 372, 774–797 (2007).

    Article  CAS  PubMed  Google Scholar 

  27. Gulick, A. M., Lu, X. & Dunaway-Mariano, D. Crystal structure of 4-chlorobenzoate:CoA ligase/synthetase in the unliganded and aryl substrate-bound states. Biochemistry 43, 8670–8679 (2004).

    Article  CAS  PubMed  Google Scholar 

  28. Osman, K. T., Du, L., He, Y. & Luo, Y. Crystal structure of Bacillus cereus d-alanyl carrier protein ligase (DltA) in complex with ATP. J. Mol. Biol. 388, 345–355 (2009).

    Article  CAS  PubMed  Google Scholar 

  29. Wu, R. et al. Mechanism of 4-chlorobenzoate:coenzyme a ligase catalysis. Biochemistry 47, 8026–8039 (2008).

    Article  CAS  PubMed  Google Scholar 

  30. Horswill, A. R. & Escalante-Semerena, J. C. Characterization of the propionyl-CoA synthetase (PrpE) enzyme of Salmonella enterica: residue Lys592 is required for propionyl-AMP synthesis. Biochemistry 41, 2379–2387 (2002).

    Article  CAS  PubMed  Google Scholar 

  31. Starai, V. J., Celic, I., Cole, R. N., Boeke, J. D. & Escalante-Semerena, J. C. Sir2-dependent activation of acetyl-CoA synthetase by deacetylation of active lysine. Science 298, 2390–2392 (2002).

    Article  CAS  PubMed  Google Scholar 

  32. Schwer, B., Bunkenborg, J., Verdin, R. O., Andersen, J. S. & Verdin, E. Reversible lysine acetylation controls the activity of the mitochondrial enzyme acetyl-CoA synthetase 2. Proc. Natl. Acad. Sci. USA 103, 10224–10229 (2006).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  33. Trivedi, O. A. et al. Enzymic activation and transfer of fatty acids as acyl-adenylates in mycobacteria. Nature 428, 441–445 (2004).

    Article  CAS  PubMed  Google Scholar 

  34. Arora, P. et al. Mechanistic and functional insights into fatty acid activation in Mycobacterium tuberculosis. Nat. Chem. Biol. 5, 166–173 (2009).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Zhang, Z. et al. Structural and functional studies of fatty acyl adenylate ligases from E. coli and L. pneumophila. J. Mol. Biol. 406, 313–324 (2011).

    Article  CAS  PubMed  Google Scholar 

  36. Goyal, A., Verma, P., Anandhakrishnan, M., Gokhale, R. S. & Sankaranarayanan, R. Molecular basis of the functional divergence of fatty acyl-AMP ligase biosynthetic enzymes of Mycobacterium tuberculosis. J. Mol. Biol. 416, 221–238 (2012).

    Article  CAS  PubMed  Google Scholar 

  37. Yonus, H. et al. Crystal structure of DltA. Implications for the reaction mechanism of non-ribosomal peptide synthetase adenylation domains. J. Biol. Chem. 283, 32484–32491 (2008).

    Article  CAS  PubMed  Google Scholar 

  38. Gulick, A. M., Starai, V. J., Horswill, A. R., Homick, K. M. & Escalante-Semerena, J. C. The 1.75 A crystal structure of acetyl-CoA synthetase bound to adenosine-5′-propylphosphate and coenzyme A. Biochemistry 42, 2866–2873 (2003).

    Article  CAS  PubMed  Google Scholar 

  39. Shi, J. et al. Titer improvement and pilot-scale production of platensimycin from Streptomyces platensis SB12026. J. Ind. Microbiol. Biotechnol. 43, 1027–1035 (2016).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  40. Stols, L., Millard, C. S., Dementieva, I. & Donnelly, M. I. Production of selenomethionine-labeled proteins in two-liter plastic bottles for structure determination. J. Struct. Funct. Genomics 5, 95–102 (2004).

    Article  CAS  PubMed  Google Scholar 

  41. Minor, W., Cymborowski, M., Otwinowski, Z. & Chruszcz, M. HKL-3000: the integration of data reduction and structure solution–from diffraction images to an initial model in minutes. Acta Crystallogr. D Biol. Crystallogr. 62, 859–866 (2006).

    Article  CAS  PubMed  Google Scholar 

  42. Terwilliger, T. C. et al. Iterative model building, structure refinement and density modification with the PHENIX AutoBuild wizard. Acta Crystallogr. D Biol. Crystallogr. 64, 61–69 (2008).

    Article  CAS  PubMed  Google Scholar 

  43. Adams, P. D. et al. PHENIX: a comprehensive Python-based system for macromolecular structure solution. Acta Crystallogr. D Biol. Crystallogr. 66, 213–221 (2010).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  44. Vagin, A. & Teplyakov, A. Molecular replacement with MOLREP. Acta Crystallogr. D Biol. Crystallogr. 66, 22–25 (2010).

    Article  CAS  PubMed  Google Scholar 

  45. Winn, M. D. et al. Overview of the CCP4 suite and current developments. Acta Crystallogr. D Biol. Crystallogr. 67, 235–242 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  46. Chen, V. B. et al. MolProbity: all-atom structure validation for macromolecular crystallography. Acta Crystallogr. D Biol. Crystallogr. 66, 12–21 (2010).

    Article  CAS  PubMed  Google Scholar 

  47. Sievers, F. et al. Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539 (2011).

    Article  PubMed  PubMed Central  Google Scholar 

  48. Tamura, K. et al. MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol. Biol. Evol. 28, 2731–2739 (2011).

    Article  CAS  PubMed  PubMed Central  Google Scholar 

Download references

Acknowledgements

This work is supported in part by the National Institute of General Medical Sciences Protein Structure Initiative grants GM094585 (A.J.) and GM098248 (G.N.P.) and National Institutes of Health grants GM109456 (G.N.P.) and GM114353 (B.S.). The use of Structural Biology Center beamlines at the Advanced Photon Source was supported by US Department of Energy, Office of Biological and Environmental Research grant DE-AC02-06CH11357 (A.J.). N.W. is supported in part by the Institute of Applied Ecology, Chinese Academy of Sciences, and a scholarship from the Chinese Scholarship Council (201504910034). J.D.R. is supported in part by an Arnold O. Beckman Postdoctoral Fellowship. C.-Y.C. is supported in part by the Fellowship of Academia Sinica–The Scripps Research Institute Postdoctoral Talent Development Program. This is manuscript #29600 from The Scripps Research Institute.

Author information

Author notes

  1. These authors contributed equally: Nan Wang, Jeffrey D. Rudolf.

Authors and Affiliations

  1. Department of Chemistry, The Scripps Research Institute, Jupiter, FL, USA

    Nan Wang, Jeffrey D. Rudolf, Liao-Bin Dong, Chin-Yuan Chang & Ben Shen

  2. Midwest Center for Structural Genomics and Structural Biology Center, Biosciences Division, Argonne National Laboratory, Argonne, IL, USA

    Jerzy Osipiuk, Catherine Hatzos-Skintges, Michael Endres, Gyorgy Babnigg & Andrzej Joachimiak

  3. Department of Biosciences, Rice University, Houston, TX, USA

    George N. Phillips Jr

  4. Department of Molecular Medicine, The Scripps Research Institute, Jupiter, FL, USA

    Ben Shen

  5. Natural Products Library Initiative at The Scripps Research Institute, The Scripps Research Institute, Jupiter, FL, USA

    Ben Shen

Authors
  1. Nan Wang
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeffrey D. Rudolf
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Liao-Bin Dong
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Jerzy Osipiuk
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Catherine Hatzos-Skintges
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Michael Endres
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Chin-Yuan Chang
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Gyorgy Babnigg
    View author publications

    You can also search for this author in PubMed Google Scholar

  9. Andrzej Joachimiak
    View author publications

    You can also search for this author in PubMed Google Scholar

  10. George N. Phillips Jr
    View author publications

    You can also search for this author in PubMed Google Scholar

  11. Ben Shen
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

B.S., G.N.P., and A.J. conceived the project; N.W., J.D.R., L.-B.D., G.N.P. and B.S. designed the experiments; N.W. and J.D.R. performed bioinformatics, molecular cloning, protein production and purification, biochemical analysis, and enzyme reactions; J.D.R. constructed the genetic knockouts in Streptomyces; N.W., J.D.R., and L.-B.D. conducted Streptomyces fermentation; N.W. and L.-B.D. performed natural product isolation and structure determination; C.H.-S. and M.E. performed crystallography; J.O. conducted protein structure determination; N.W., J.D.R., L.-B.D., J.O., C.H.-S., M.E., C.-Y.C., G.B., and B.S. analyzed the results; N.W. and J.D.R. wrote the first draft with input from all co-authors; J.D.R. and B.S. revised and finalized the manuscript.

Corresponding author

Correspondence to Ben Shen.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Text and Figures

Supplementary Tables 1–4, Supplementary Figures 1–17

Reporting Summary

Supplementary Note

Spectroscopic data for diterpenoids 3–10

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Wang, N., Rudolf, J.D., Dong, LB. et al. Natural separation of the acyl-CoA ligase reaction results in a non-adenylating enzyme. Nat Chem Biol 14, 730–737 (2018). https://doi.org/10.1038/s41589-018-0061-0

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/s41589-018-0061-0

This article is cited by

Search

Advanced search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing