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

William A. Barton; Jacob Lesniak; John B. Biggins; Philip D. Jeffrey; Jiqing Jiang; K. R. Rajashankar; Jon S. Thorson; Dimitar B. Nikolov

doi:10.1038/88618

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

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

Research output: Contribution to journal › Article › peer-review

121 Scopus citations

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 (E_p) from salmonella, in complex with product (udp-gic) and substrate (dttp) is reported at 2.0 Å and 2.1 Å resolution, respectively. These structures, in conjunction with the kinetic characterization of E_p, clarify the catalytic mechanism of this important enzyme class. Structure-based engineering of E_p produced modified enzymes capable of utilizing 'unnatural' sugar phosphates not accepted by wild type E_p. 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.

Original language	English
Pages (from-to)	545-551
Number of pages	7
Journal	Nature Structural Biology
Volume	8
Issue number	6
DOIs	https://doi.org/10.1038/88618
State	Published - 2001

Bibliographical note

Funding Information:
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.

ASJC Scopus subject areas

Structural Biology
Biochemistry
Genetics

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

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-gic) and substrate (dttp) is reported at 2.0 {\AA} and 2.1 {\AA} 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.",

author = "Barton, {William A.} and Jacob Lesniak and Biggins, {John B.} and Jeffrey, {Philip D.} and Jiqing Jiang and Rajashankar, {K. R.} and Thorson, {Jon S.} and Nikolov, {Dimitar B.}",

note = "Funding Information: 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.",

year = "2001",

doi = "10.1038/88618",

language = "English",

volume = "8",

pages = "545--551",

number = "6",

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T1 - Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization

AU - Barton, William A.

AU - Lesniak, Jacob

AU - Biggins, John B.

AU - Jeffrey, Philip D.

AU - Jiang, Jiqing

AU - Rajashankar, K. R.

AU - Thorson, Jon S.

AU - Nikolov, Dimitar B.

N1 - Funding Information: 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.

PY - 2001

Y1 - 2001

N2 - 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-gic) 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.

AB - 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-gic) 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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Structure, mechanism and engineering of a nucleotidylyltransferase as a first step toward glycorandomization

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