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Engineering the product profile of a polysialyltransferase

Nature Chemical Biology volume 10, pages 437–442 (2014)Cite this article

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Abstract

Oligo- and polysaccharides have myriad applications as therapeutic reagents from glycoconjugate vaccines to matrices for tissue engineering. Polysaccharide length may vary over several orders of magnitude and is a critical determinant of both their physical properties and biological activities. Therefore, the tailored synthesis of oligo- and polysaccharides of defined size is a major goal for glycoengineering. By mutagenesis and screening of a bacterial polysialyltransferase (polyST), we identified a single-residue switch that controls the size distribution of polymeric products. Specific substitutions at this site yielded distributive enzymes that synthesize polysaccharides with narrow size distribution ideal for glycoengineering applications. Mechanistic investigation revealed that the wild-type enzyme has an extended binding site that accommodates at least 20 residues of the growing polymer; changes in affinity along this binding site allow fine-tuning of the enzyme's product distribution.

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Figure 1: Mechanisms of polysaccharide chain elongation and schematic HPLC product profiles.
Figure 2: Drifted polySTs exhibit diverse patterns of chain elongation.
Figure 3: Saturated mutagenesis clones trace a limit for low product dispersity.
Figure 4: PolyST product distribution is determined by acceptor preference.
Figure 5: Single amino acid exchanges which alter the polyST product distribution.

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Acknowledgements

We thank M. Berger and A. Bethe for expert technical assistance. This work was supported by the Deutsche Forschungsgemeinschaft (DFG) in the framework of DFG Research Unit 548 to R.G.-S. (grant no. GE 801/6-2 and Ge801/10-1).

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Authors and Affiliations

  1. Institute for Cellular Chemistry, Hannover Medical School, Hannover, Germany

    Timothy G Keys, Hazel L S Fuchs, Jörg Ehrit, Friedrich Freiberger & Rita Gerardy-Schahn

  2. Institute for Biophysical Chemistry, Hannover Medical School, Hannover, Germany

    Jürgen Alves

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  1. Timothy G Keys
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Contributions

T.G.K. designed research, performed experiments, analyzed the data and wrote the manuscript. J.E. performed some in vivo screening and contributed to the kinetic analysis and preparation of DMB-oligoSia. H.L.S.F. prepared DNA constructs for single-mutant analysis. J.A. supervised the collection and analysis of CD spectra. F.F. and R.G.-S. provided guidance on experimental design and interpretation of results. R.G.-S. supervised the project and contributed to writing the manuscript. All of the authors discussed the results and gave feedback on the manuscript.

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Correspondence to Rita Gerardy-Schahn.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Results, Supplementary Tables 1–3 and Supplementary Figures 1–10. (PDF 1988 kb)

Supplementary Data Set 1

Part 1 – High-dispersity clones. Part 2 – Medium-dispersity clones. Part 3 – Low-dispersity clones. Part 4 – Reference clones. (PDF 2837 kb)

Supplementary Data Set 2

The product profiles of enzymes in the low dispersity category closely fit a Poisson distribution. (PDF 3551 kb)

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Keys, T., Fuchs, H., Ehrit, J. et al. Engineering the product profile of a polysialyltransferase. Nat Chem Biol 10, 437–442 (2014). https://doi.org/10.1038/nchembio.1501

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