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Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization

Nature Structural Biology volume 8, pages 545–551 (2001)Cite this article

Abstract

Metabolite glycosylation is affected by three classes of enzymes: nucleotidylyltransferases, which activate sugars as nucleotide diphospho-derivatives, intermediate sugar-modifying enzymes and glycosyltransferases, which transfer the final derivatized activated sugars to aglycon substrates. One of the first crystal structures of an enzyme responsible for the first step in this cascade, α-D-glucopyranosyl phosphate thymidylyltransferase (Ep) from Salmonella, in complex with product (UDP-Glc) and substrate (dTTP) is reported at 2.0 Å and 2.1 Å resolution, respectively. These structures, in conjunction with the kinetic characterization of Ep, clarify the catalytic mechanism of this important enzyme class. Structure-based engineering of Ep produced modified enzymes capable of utilizing 'unnatural' sugar phosphates not accepted by wild type Ep. The demonstrated ability to alter nucleotidylyltransferase specificity by design is an integral component of in vitro glycosylation systems developed for the production of diverse glycorandomized libraries.

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Figure 1: Potential of Ep for glycosylation.
Figure 2: Structure of Ep bound to UDP-Glc or dTTP.
Figure 3: The Ep active site.
Figure 4: Ep catalytic mechanism.

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Acknowledgements

This contribution was supported in part by the National Institutes of Health to D.B.N. and J.S.T., a Cancer Center Support Grant and a grant from the Special Projects Committee of the Society of Memorial Sloan-Kettering Cancer Center. D.B.N. is a Pew Scholar. J.S.T. is an Alfred P. Sloan Research Fellow and a Rita Allen Foundation Scholar.

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Authors and Affiliations

  1. Cellular Biochemistry and Biophysics Program, Joan and Sanford I. Weill Graduate School of Medical Sciences, Cornell University, 1275 York Avenue, New York, 10021, USA

    William A. Barton, Jacob Lesniak, Philip D. Jeffrey & Dimitar B. Nikolov

  2. Laboratory for Biosynthetic Chemistry, Joan and Sanford I. Weill Graduate School of Medical Sciences, Cornell University, 1275 York Avenue, New York, 10021, USA

    John B. Biggins, Jiqing Jiang & Jon S. Thorson

  3. Memorial Sloan-Kettering Cancer Center and the Sloan-Kettering Division, Joan and Sanford I. Weill Graduate School of Medical Sciences, Cornell University, 1275 York Avenue, New York, 10021, USA

    William A. Barton, Jacob Lesniak, John B. Biggins, Philip D. Jeffrey, Jiqing Jiang, Jon S. Thorson & Dimitar B. Nikolov

  4. Brookhaven National Laboratory, Brookhaven, 11973, New York, USA

    K. R. Rajashankar

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Correspondence to Jon S. Thorson or Dimitar B. Nikolov.

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Barton, W., Lesniak, J., Biggins, J. et al. Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization. Nat Struct Mol Biol 8, 545–551 (2001). https://doi.org/10.1038/88618

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