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Metamorphic enzyme assembly in polyketide diversification

Nature volume 459, pages 731–735 (2009)Cite this article

Abstract

Natural product chemical diversity is fuelled by the emergence and ongoing evolution of biosynthetic pathways in secondary metabolism1. However, co-evolution of enzymes for metabolic diversification is not well understood, especially at the biochemical level. Here, two parallel assemblies with an extraordinarily high sequence identity from Lyngbya majuscula form a β-branched cyclopropane in the curacin A pathway (Cur), and a vinyl chloride group in the jamaicamide pathway (Jam). The components include a halogenase, a 3-hydroxy-3-methylglutaryl enzyme cassette for polyketide β-branching, and an enoyl reductase domain. The halogenase from CurA, and the dehydratases (ECH1s), decarboxylases (ECH2s) and enoyl reductase domains from both Cur and Jam, were assessed biochemically to determine the mechanisms of cyclopropane and vinyl chloride formation. Unexpectedly, the polyketide β-branching pathway was modified by introduction of a γ-chlorination step on (S)-3-hydroxy-3-methylglutaryl mediated by Cur halogenase, a non-haem Fe(ii), α-ketoglutarate-dependent enzyme2. In a divergent scheme, Cur ECH2 was found to catalyse formation of the α,β enoyl thioester, whereas Jam ECH2 formed a vinyl chloride moiety by selectively generating the corresponding β,γ enoyl thioester of the 3-methyl-4-chloroglutaconyl decarboxylation product. Finally, the enoyl reductase domain of CurF specifically catalysed an unprecedented cyclopropanation on the chlorinated product of Cur ECH2 instead of the canonical α,β C = C saturation reaction. Thus, the combination of chlorination and polyketide β-branching, coupled with mechanistic diversification of ECH2 and enoyl reductase, leads to the formation of cyclopropane and vinyl chloride moieties. These results reveal a parallel interplay of evolutionary events in multienzyme systems leading to functional group diversity in secondary metabolites.

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Figure 1: Comparison of enzyme assemblies in the Cur and Jam pathways.
Figure 2: Halogenation and cyclopropanation in the Cur pathway.
Figure 3: Comparison of ECH 2 s and ERs in Cur and Jam pathways.
Figure 4: Impact of enzyme assembly evolution on β-branching chemical diversity.

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Acknowledgements

We thank C. T. Walsh and C. T. Calderone for ACP constructs; S. M. Chernyak, H. Liu and J. Byun for mass spectrometry assistance; P. C. López for NMR assistance; T. M. Ramsey for chiral cyclopronanecarboxylic acid; and D. L. Akey for discussions. This work was supported by grants from the National Institutes of Health (to D.H.S. and J.L.S.), a graduate fellowship from Eli Lilly & Co. and a Rackham Predoctoral Fellowship (to L.G.).

Author Contributions L.G., W.H.G and D.H.S. designed the experiments, analysed data and wrote the paper; L.G. performed the experiments; B.W. and K.H. recorded FTICR mass spectra and analysed the data; T.W.G. and J.L.S. modelled Cur ECH2 structure with the chlorinated substrate and designed site mutagenesis; A.K. and P.W. synthesized the chlorinated butylamide derivatives; R.V.G. and L.G. made Jam ECH1 and ECH2 constructs; W.H.G. provided DNA of Jam enzymes and analysed NMR data for isotope-labelled curacin A.

Author information

Author notes

  1. Amol Kulkarni and Todd W. Geders: These authors contributed equally to this work.

Authors and Affiliations

  1. Life Sciences Institute,,

    Liangcai Gu, Todd W. Geders, Janet L. Smith & David H. Sherman

  2. Department of Medicinal Chemistry,,

    Liangcai Gu & David H. Sherman

  3. Department of Chemistry,,

    Bo Wang, Kristina Håkansson & David H. Sherman

  4. Department of Biological Chemistry,,

    Janet L. Smith

  5. Department of Microbiology & Immunology, University of Michigan, Ann Arbor, Michigan 48109, USA,

    David H. Sherman

  6. Department of Chemistry and Center for Chemical Methodologies & Library Development, University of Pittsburgh, Pittsburgh, Pennsylvania 15260, USA,

    Amol Kulkarni & Peter Wipf

  7. Scripps Institution of Oceanography & Skaggs School of Pharmacy and Pharmaceutical Sciences, University of California at San Diego, La Jolla, California 92093, USA ,

    Rashel V. Grindberg, Lena Gerwick & William H. Gerwick

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Correspondence to David H. Sherman.

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Gu, L., Wang, B., Kulkarni, A. et al. Metamorphic enzyme assembly in polyketide diversification. Nature 459, 731–735 (2009). https://doi.org/10.1038/nature07870

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