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Biosynthesis of the tunicamycin antibiotics proceeds via unique exo-glycal intermediates

Nature Chemistry volume 4, pages 539–546 (2012)Cite this article

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Abstract

The tunicamycins are archetypal nucleoside antibiotics targeting bacterial peptidoglycan biosynthesis and eukaryotic protein N-glycosylation. Understanding the biosynthesis of their unusual carbon framework may lead to variants with improved selectivity. Here, we demonstrate in vitro recapitulation of key sugar-manipulating enzymes from this pathway. TunA is found to exhibit unusual regioselectivity in the reduction of a key α,β-unsaturated ketone. The product of this reaction is shown to be the preferred substrate for TunF—an epimerase that converts the glucose derivative to a galactose. In Streptomyces strains in which another gene (tunB) is deleted, the biosynthesis is shown to stall at this exo-glycal product. These investigations confirm the combined TunA/F activity and delineate the ordering of events in the metabolic pathway. This is the first time these surprising exo-glycal intermediates have been seen in biology. They suggest that construction of the aminodialdose core of tunicamycin exploits their enol ether motif in a mode of C–C bond formation not previously observed in nature, to create an 11-carbon chain.

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Figure 1: Structures of the tunicamycins and early stages of the proposed biosynthetic pathway that creates the 11-carbon chain.
Figure 2: Possible TunA-catalysed dehydration pathways for substrate UDP-GlcNAc and putative intermediates.
Figure 3: Three-dimensional structure of TunA.
Figure 4: Comparison of the conformations of substrates and cofactors in active sites between TunA and RmlB.
Figure 5: Interconversion of UDP-GlcNAc and related biosynthetic intermediates by TunA and TunF.
Figure 6: Suggested revised biosynthetic pathway to tunicamine, a key precursor of tunicamycin.

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Acknowledgements

The authors gratefully acknowledge B. Odell (Oxford University) for help with the NMR experiments. This work was supported by the EPSRC (DTA studentship for F.J.W.), the Bill and Melinda Gates Foundation (S.S.L.) and BBSRC (J.P.G-E. and M.J.B). G.J.D and B.G.D. are Royal Society Wolfson Research Merit Award recipients. This manuscript is dedicated to the memory of Professor David Gin.

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  1. Filip J. Wyszynski and Seung Seo Lee: These authors contributed equally to this work

Authors and Affiliations

  1. Chemistry Research Laboratory, University of Oxford, 12 Mansfield Road, Oxford, OX1 3TA, UK

    Filip J. Wyszynski, Seung Seo Lee, Tomoaki Yabe, Hua Wang & Benjamin G. Davis

  2. Department of Molecular Microbiology, John Innes Centre, Norwich Research Park, Norwich, NR4 7UH, UK

    Juan Pablo Gomez-Escribano & Mervyn J. Bibb

  3. College of Pharmacy, Chungbuk National University, Gaeshin-dong, Heungduk-gu, Cheongju, Chungbuk, Korea

    Soo Jae Lee

  4. Department of Chemistry, The University of York, Heslington, YO10 5DD, York, UK

    Gideon J. Davies

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Contributions

F.J.W. cloned tunF, purified the protein and performed functional and kinetic analyses of TunF. S.S.L. and T.Y. cloned tunA and its mutants, purified the resulting proteins and performed functional analyses. S.S.L. performed the kinetic analyses. TunA was crystallized by T.Y. The 3D structure was determined by S.J.L., and S.S.L., B.G.D and G.J.D. carried out its analysis. H.W. and J.P.G-E. created the tunB mutant strain and extracts. M.J.B. designed the deletion strategy. H.W., M.J.B. and B.G.D. analysed the extracts. The manuscript was written by F.J.W., S.S.L., M.J.B., G.J.D. and B.G.D.

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Correspondence to Benjamin G. Davis.

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The University of Oxford has filed a patent, in which the authors are named as inventors, on the utility of the biosynthetic cluster and enzymes derived from it.

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Wyszynski, F., Lee, S., Yabe, T. et al. Biosynthesis of the tunicamycin antibiotics proceeds via unique exo-glycal intermediates. Nature Chem 4, 539–546 (2012). https://doi.org/10.1038/nchem.1351

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