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Structural dissection and high-throughput screening of mannosylglycerate synthase

Nature Structural & Molecular Biology volume 12, pages 608–614 (2005)Cite this article

Abstract

The enzymatic transfer of activated mannose yields mannosides in glycoconjugates and oligo- and polysaccharides. Yet, despite its biological necessity, the mechanism by which glycosyltransferases recognize mannose and catalyze its transfer to acceptor molecules is poorly understood. Here, we report broad high-throughput screening and kinetic analyses of both natural and synthetic substrates of Rhodothermus marinus mannosylglycerate synthase (MGS), which catalyzes the formation of the stress protectant 2-O-α-D-mannosyl glycerate. The sequence of MGS indicates that it is at the cusp of inverting and retaining transferases. The structures of apo MGS and complexes with donor and acceptor molecules, including GDP-mannose, combined with mutagenesis of the binding and catalytic sites, unveil the mannosyl transfer center. Nucleotide specificity is as important in GDP-D-mannose recognition as the nature of the donor sugar.

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Figure 1: High-throughput screening of GT specificity.
Figure 2: Three-dimensional structure of MGS.
Figure 3: Observed electron density for MGS ligand complexes.
Figure 4: Comparison of GT-A fold retaining and inverting GTs.
Figure 5: A 'front-face' mechanism for glycosyl transfer with retention of anomeric configuration.

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Acknowledgements

The authors thank G. Sheldrick (Göttingen) for assistance with SHELXD. This work was funded by Biotechnology and Biological Sciences Research Council and the Wellcome Trust. G.J.D. is a Royal Society University Research Fellow.

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Authors and Affiliations

  1. Institute for Cell and Molecular Biosciences, University of Newcastle upon Tyne, The Medical School, Newcastle upon Tyne, NE2 4HH, UK

    James Flint, David N Bolam, Louise E Tailford & Harry J Gilbert

  2. Department of Chemistry, York Structural Biology Laboratory, University of York, York, Y010 5YW, UK

    Edward Taylor, Carlos Martinez-Fleites, Eleanor J Dodson & Gideon J Davies

  3. Department of Chemistry, The University of Oxford, Mansfield Road, Oxford, OX1 3TA, UK

    Min Yang & Benjamin G Davis

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  1. James Flint
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Correspondence to Gideon J Davies.

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Supplementary information

Supplementary Fig. 1

Compounds in the GAR broad screen acceptor library. (PDF 95 kb)

Supplementary Methods (PDF 128 kb)

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Flint, J., Taylor, E., Yang, M. et al. Structural dissection and high-throughput screening of mannosylglycerate synthase. Nat Struct Mol Biol 12, 608–614 (2005). https://doi.org/10.1038/nsmb950

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