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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.

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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.

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

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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.

Competing interests

The authors declare no competing interests.

Corresponding author

Correspondence to Ben Shen.

Supplementary information

  1. Supplementary Text and Figures

    Supplementary Tables 1–4, Supplementary Figures 1–17

  2. Reporting Summary

  3. Supplementary Note

    Spectroscopic data for diterpenoids 3–10

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DOI

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