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Designer enzymes for glycosphingolipid synthesis by directed evolution

Nature Chemical Biology volume 5, pages 508–514 (2009)Cite this article

Abstract

Though glycosphingolipids have great potential as therapeutics for cancer, HIV, neurodegenerative diseases and auto-immune diseases, both extensive study of their biological roles and development as pharmaceuticals are limited by difficulties in their synthesis, especially on large scales. Here we addressed this restriction by expanding the synthetic scope of a glycosphingolipid-synthesizing enzyme through a combination of rational mutagenesis and directed evolution with an ELISA-based screening strategy. We targeted both a low-level promiscuous substrate activity and the overall catalytic efficiency of the catalyst, and we identified several mutants with enhanced activities. These new catalysts, which are capable of producing a broad range of homogeneous samples, represent a significant advance toward the facile, large-scale synthesis of glycosphingolipids and demonstrate the general utility of this approach toward the creation of designer glycosphingolipid-synthesizing enzymes.

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Figure 1: The synthesis of glycosphingolipids by EGC glycosynthase.
Figure 2: The structural location of the identified glycosynthase mutants.

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Acknowledgements

The authors thank the Royal Society (UK) and the Government of Canada for postdoctoral fellowships (S.M.H.), the Natural Sciences and Engineering Research Council of Canada and Neose Technologies Ltd. for funding, and W. Hol (University of Washington) for providing the recombinant gene for cholera toxin B subunit.

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Authors and Affiliations

  1. Department of Chemistry and Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, British Columbia, Canada

    Susan M Hancock, Jamie R Rich, Matthew E C Caines, Natalie C J Strynadka & Stephen G Withers

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  1. Susan M Hancock
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  2. Jamie R Rich
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  3. Matthew E C Caines
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  4. Natalie C J Strynadka
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  5. Stephen G Withers
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Contributions

S.M.H. and S.G.W. designed the experiments; S.M.H. performed the experiments; S.M.H., M.E.C.C. and S.G.W. analyzed the data; J.R.R. characterized the synthetic products; S.M.H. and S.G.W. wrote the manuscript; M.E.C.C. and S.M.H. made the figures; M.E.C.C. and N.C.J.S. provided feedback on the manuscript.

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Correspondence to Stephen G Withers.

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The authors have requested patent protection on the results of this study through the University of British Columbia, who will own the patent.

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Supplementary Figures 1–6, Supplementary Tables 1–3 and Supplementary Methods (PDF 3278 kb)

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Hancock, S., Rich, J., Caines, M. et al. Designer enzymes for glycosphingolipid synthesis by directed evolution. Nat Chem Biol 5, 508–514 (2009). https://doi.org/10.1038/nchembio.191

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