Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis
Broadening the scope of glycosyltransferase-catalyzed sugar nucleotide synthesis
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DOI：
10.1073/pnas.1220220110
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发表时间：
2013-05-07
影响因子：
11.1
通讯作者：
Thorson, Jon S.
中科院分区：
文献类型：
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作者：
Gantt, Richard W.;Peltier-Pain, Pauline;Thorson, Jon S.
我们描述了糖基转移酶催化反应的一般可逆性、人工糖基供体和高通量比色筛选的整合，以使糖基转移酶的工程化能够用于组合糖核苷酸合成。与OleD野生型序列相比，来自本研究的最佳工程化催化剂OleD Loki变体包含突变P67 T/I112 P/T113 M/S132 F/A242 I。相对于用于筛选的亲本序列OleD TDP 16变体进行评估，OleD Loki变体对于NDP-葡萄糖和UDP-糖的形成分别显示出> 400倍和> 15倍的k（cat）/K-m的最大改善。这种OleD Loki变体还证明了与五种变体NDP受体和六种变体2-氯-4-硝基苯基糖苷供体的有效周转，以产生30种不同的NDP-糖。这项研究突出了一个方便的策略，以快速优化糖基转移酶催化剂的合成复杂的糖核苷酸和一套独特的糖核苷酸的实际合成。
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.