TY - JOUR
T1 - Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis
AU - Gantt, Richard W.
AU - Peltier-Pain, Pauline
AU - Singh, Shanteri
AU - Zhou, Maoquan
AU - Thorson, Jon S.
PY - 2013/5/7
Y1 - 2013/5/7
N2 - We described the integration of the general reversibility of glycosyltransferase- catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k cat/Km of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2- chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.
AB - We described the integration of the general reversibility of glycosyltransferase- catalyzed reactions, artificial glycosyl donors, and a high throughput colorimetric screen to enable the engineering of glycosyltransferases for combinatorial sugar nucleotide synthesis. The best engineered catalyst from this study, the OleD Loki variant, contained the mutations P67T/I112P/T113M/S132F/A242I compared with the OleD wild-type sequence. Evaluated against the parental sequence OleD TDP16 variant used for screening, the OleD Loki variant displayed maximum improvements in k cat/Km of >400-fold and >15-fold for formation of NDP-glucoses and UDP-sugars, respectively. This OleD Loki variant also demonstrated efficient turnover with five variant NDP acceptors and six variant 2- chloro-4-nitrophenyl glycoside donors to produce 30 distinct NDP-sugars. This study highlights a convenient strategy to rapidly optimize glycosyltransferase catalysts for the synthesis of complex sugar nucleotides and the practical synthesis of a unique set of sugar nucleotides.
KW - Carbohydrate
KW - Enzyme
KW - Glycobiology
KW - Protein engineering
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U2 - 10.1073/pnas.1220220110
DO - 10.1073/pnas.1220220110
M3 - Article
C2 - 23610417
AN - SCOPUS:84877359433
SN - 0027-8424
VL - 110
SP - 7648
EP - 7653
JO - Proceedings of the National Academy of Sciences of the United States of America
JF - Proceedings of the National Academy of Sciences of the United States of America
IS - 19
ER -