Engineering ribonucleoside triphosphate specificity in a thymidylyltransferase
Engineering ribonucleoside triphosphate specificity in a thymidylyltransferase
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DOI：
10.1021/bi800978u
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发表时间：
2008-08-19
期刊：
影响因子：
2.9
通讯作者：
Ferrieres, Vincent
中科院分区：
文献类型：
--
作者：
Jakeman, David L.;Young, Jessica L.;Ferrieres, Vincent
自然界的糖基化催化剂，糖基转移酶，通过将糖核苷酸供体转移到受体上间接操纵和控制许多重要的生物过程。具有挑战性的化学合成阻碍了对糖核苷酸的合成，限制了许多糖基转移酶的研究。酶对糖核苷酸的获取是一个迅速发展的研究途径，仅受酶的底物特异性的限制。我们探索了从肺炎链球菌中分离出来的混杂胸腺基转移酶Cps2L，并通过活性位点工程增强了其尿苷基转移酶和胍苷基转移酶的活性。在Q24位点突变导致了一个变异，udp -氨基葡萄糖、udp -甘露糖和udp - n -乙酰氨基葡萄糖的产量分别增加了10倍、3倍和2倍。Cps2L变体在野生型酶上观察到新的催化活性，包括gdp -甘露糖的形成。该变体被评价为形成一系列dTDP-和udp -呋喃酶的催化剂，并在12小时后以90%的产率产生dTDP- galf，以30%的产率产生UDP-Araf。一系列3- o -烷基葡萄糖i -磷酸也被评价为底物，并显著转化为udp -3- o -甲基葡萄糖和udp -3- o -十二烷基葡萄糖，而野生型酶则没有。与野生型酶相比，Q24S变体基本上也增强了所有胸苷基转移酶的活性。对核苷酸基转移酶和胸腺基转移酶活性位点与产物结合的比较表明，Q24S突变体是扩大核苷酸基转移酶活性的新途径。
Nature's glycosylation catalysts, glycosyltransferases, indirectly manipulate and control many important biological processes by transferring sugar nucleotide donors onto acceptors. Challenging chemical synthesis impedes synthetic access to sugar nucleotides and limits the study of many glycosyltransferases. Enzymatic access to sugar nucleotides is a rapidly expanding avenue of research, limited only by the substrate specificity of the enzyme. We have explored the promiscuous thymidylyltransferase from Streptococcus pneumoniae, Cps2L, and enhanced its uridylyltransferase and guanidyltransferase activities by active site engineering. Mutagenesis at position Q24 resulted in a variant with 10-, 3-, and 2-fold enhancement of UDP-glucosamine, UDP-mannose, and UDP-N-acetylglucosamine production, respectively. New catalytic activities were observed for the Cps2L variant over the wild-type enzyme, including the formation of GDP-mannose. The variant was evaluated as a catalyst for the formation of a series of dTDP- and UDP-furanoses and notably produced dTDP-Galf in 90% yield and UDP-Araf in 30% yield after 12 h. A series of 3-O-alkylglucose I-phosphates were also evaluated as substrates, and notable conversions to UDP-3-O-methylglucose and UDP-3-O-dodecylglucose were achieved with the variant but not the wild-type enzyme. The Q24S variant also enhanced essentially all thymidylyltransferase activities relative to the wild-type enzyme. Comparison of active sites of uridylyltransferases and thymidylyltransferases with products bound indicate the Q24S variant to be a new approach in broadening nucleotidylyltransferase activity.