Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis

被引：87

作者：

Gantt, Richard W. ^{[1
]}

Peltier-Pain, Pauline ^{[1
]}

Singh, Shanteri ^{[2
]}

Zhou, Maoquan ^{[1
]}

Thorson, Jon S. ^{[2
]}

机构：

[1] Univ Wisconsin, Sch Pharm, Wisconsin Ctr Nat Prod Res, Pharmaceut Sci Div, Madison, WI 53705 USA

[2] Univ Kentucky, Coll Pharm, Ctr Pharmaceut Res & Innovat, Lexington, KY 40536 USA

来源：

PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA | 2013年 / 110卷 / 19期

基金：

美国国家卫生研究院;

关键词：

carbohydrate; enzyme; glycobiology; protein engineering; NATURAL-PRODUCT GLYCOSYLTRANSFERASE; BIOSYNTHETIC-PATHWAY; CRYSTAL-STRUCTURE; GLYCOSYLATION; ENZYMES; GLUCOSYLTRANSFERASE; POLY(ADP-RIBOSE); REVERSIBILITY; PROMISCUITY; DEOXYSUGARS;

D O I：

10.1073/pnas.1220220110

中图分类号：

O [数理科学和化学]; P [天文学、地球科学]; Q [生物科学]; N [自然科学总论];

学科分类号：

07 ; 0710 ; 09 ;

摘要：

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)/K-m 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.

引用

页码：7648 / 7653

页数：6

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