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Using substrate engineering to harness enzymatic promiscuity and expand biological catalysis

Nature Chemical Biology volume 2, pages 724–728 (2006)Cite this article

Abstract

Despite their unparalleled catalytic prowess and environmental compatibility, enzymes have yet to see widespread application in synthetic chemistry. This lack of application and the resulting underuse of their enormous potential stems not only from a wariness about aqueous biological catalysis on the part of the typical synthetic chemist but also from limitations on enzyme applicability that arise from the high degree of substrate specificity possessed by most enzymes. This latter perceived limitation is being successfully challenged through rational protein engineering1,2 and directed evolution efforts3,4,5,6 to alter substrate specificity. However, such programs require considerable effort to establish. Here we report an alternative strategy for expanding the substrate specificity, and therefore the synthetic utility, of a given enzyme through a process of 'substrate engineering'. The attachment of a readily removable functional group to an alternative glycosyltransferase substrate induces a productive binding mode, facilitating rational control of substrate specificity and regioselectivity using wild-type enzymes.

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Figure 1
Figure 2: Substrate engineering strategy applied to various alternative LgtC acceptor substrates.
Figure 3: A model for the substrate-engineering strategy using 6-OBz Man.

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Acknowledgements

This work was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC), the Canadian Institutes for Health Research (CIHR) and the Protein Engineering Network of Centres of Excellence of Canada (PENCE). L.L.L is the recipient of a Michael Smith Foundation for Health Research (MSFHR) Senior Graduate Studentship and a NSERC doctoral postgraduate scholarship.

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Authors and Affiliations

  1. Department of Chemistry, University of British Columbia, Vancouver, V6T 1Z1, British Columbia, Canada

    Luke L Lairson & Stephen G Withers

  2. Department of Pharmacy and Pharmacology, University of Bath, Bath, BA2 7AY, UK

    Andrew G Watts

  3. Institute of Biological Sciences, National Research Council of Canada, Ottawa, K1A 0R6, Ontario, Canada

    Warren W Wakarchuk

  4. Department of Biochemistry and Molecular Biology, University of British Columbia, Vancouver, V6T 1Z3, British Columbia, Canada

    Stephen G Withers

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  1. Luke L Lairson
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  4. Stephen G Withers
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Contributions

L.L. and A.W. conceived of this project, which was refined with input from S.W. and W.W. L.L. performed essentially all of the experimental work and, in conjunction with S.W., wrote the manuscript with input from A.W. and W.W.

Corresponding author

Correspondence to Stephen G Withers.

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Supplementary information

Supplementary Fig. 1

1H-COSY NMR spectra of 19. (PDF 205 kb)

Supplementary Fig. 2

Alternative CSt II acceptor substrates. (PDF 92 kb)

Supplementary Fig. 3

1H-COSY NMR spectra of 22. (PDF 197 kb)

Supplementary Fig. 4

1H-COSY NMR spectra of 23. (PDF 207 kb)

Supplementary Fig. 5

1H-COSY NMR spectra of 24. (PDF 182 kb)

Supplementary Fig. 6

1H-COSY NMR spectra of 28. (PDF 98 kb)

Supplementary Fig. 7

1H-COSY NMR spectra of 29. (PDF 137 kb)

Supplementary Methods (PDF 35 kb)

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Lairson, L., Watts, A., Wakarchuk, W. et al. Using substrate engineering to harness enzymatic promiscuity and expand biological catalysis. Nat Chem Biol 2, 724–728 (2006). https://doi.org/10.1038/nchembio828

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