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Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution

Nature Chemical Biology volume 3, pages 657–662 (2007)Cite this article

Abstract

Natural products, many of which are decorated with essential sugar residues, continue to serve as a key platform for drug development1. Adding or changing sugars attached to such natural products can improve the parent compound's pharmacological properties, specificity at multiple levels2, and/or even the molecular mechanism of action3. Though some natural-product glycosyltransferases (GTs) are sufficiently promiscuous for use in altering these glycosylation patterns, the stringent specificity of others remains a limiting factor in natural-product diversification and highlights a need for general GT engineering and evolution platforms. Herein we report the use of a simple high-throughput screen based on a fluorescent surrogate acceptor substrate to expand the promiscuity of a natural-product GT via directed evolution. Cumulatively, this study presents variant GTs for the glycorandomization of a range of therapeutically important acceptors, including aminocoumarins, flavonoids and macrolides, and a potential template for engineering other natural-product GTs.

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Figure 1: Enzymatic glycosylation.
Figure 2: Outcome of OleD directed evolution.
Figure 3: Activity of wild-type OleD and variant P67T/A242V/S132F toward a set of NDP-sugar donors.
Figure 4: Location of functional amino acid mutations.

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GenBank/EMBL/DDBJ

Protein Data Bank

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Acknowledgements

We are grateful to the School of Pharmacy Analytical Instrumentation Center for analytical support, H.-W. Liu (University of Texas-Austin) for plasmid pET28/OleD and S. Singh for helpful discussions. This work was supported in part by the US National Institutes of Health grants AI52218 and U19 CA113297. J.S.T. is a University of Wisconsin H.I. Romnes Fellow.

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  1. Pharmaceutical Sciences Division, Laboratory for Biosynthetic Chemistry, School of Pharmacy, National Cooperative Drug Discovery Program, University of Wisconsin-Madison, 777 Highland Avenue, Madison, 53705, Wisconsin, USA

    Gavin J Williams, Changsheng Zhang & Jon S Thorson

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  1. Gavin J Williams
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Contributions

G.J.W. contributed to the experimental design, experimental execution and manuscript drafting; C.Z. contributed experimental reagents and consultation; and J.S.T. contributed to the experimental design and manuscript drafting.

Corresponding author

Correspondence to Jon S Thorson.

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J.S.T. is a founding scientist of Centrose, LLC.

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Supplementary Figures 1–5, Supplementary Tables 1 and 2, Supplementary Methods (PDF 549 kb)

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Williams, G., Zhang, C. & Thorson, J. Expanding the promiscuity of a natural-product glycosyltransferase by directed evolution. Nat Chem Biol 3, 657–662 (2007). https://doi.org/10.1038/nchembio.2007.28

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