Chemoenzymatic synthesis of an isoprenoid phosphate tool for the analysis of complex bacterial oligosaccharide biosynthesis
Chemoenzymatic synthesis of an isoprenoid phosphate tool for the analysis of complex bacterial oligosaccharide biosynthesis
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DOI：
10.1016/j.carres.2012.06.014
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发表时间：
2012-10-01
影响因子：
3.1
通讯作者：
Troutman, Jerry M.
中科院分区：
文献类型：
--
作者：
Lujan, Donovan K.;Stanziale, Jennifer A.;Troutman, Jerry M.
十一异戊二烯焦磷酸合酶（UPPS）是催化细菌戊烯醇产生的关键酶，细菌戊烯醇作为膜锚用于组装复杂的细菌寡糖。理解寡糖组装的主要障碍之一是缺乏研究该过程的化学工具，因为bactoprenols和所得的类异戊二烯连接的寡糖缺乏用于途径分析的手柄或发色团。在这里，我们描述了从共生微生物脆弱拟杆菌（Bacteroides fragilis）中分离出一种新的UPPS，脆弱拟杆菌是人类微生物组中的一个关键物种。将蛋白质纯化至均一，并用于接受含有法呢基二磷酸类似物的发色团作为底物。该类似物被酶利用，并产生一种bactoprenyl二磷酸产物，该产物具有易于监测的标记。此外，使用普通分子生物学试剂，二磷酸被证明很容易转化为单磷酸。该单磷酸产物允许复杂寡糖生物合成的研究，并用于探测参与良好表征的空肠弯曲菌N-连接蛋白糖基化的糖基转移酶的活性。类似于此的新型试剂将为未表征的寡糖组装体的研究提供关键工具，并为这些生物合成系统的快速筛选方法的开发开辟了可能性。(c)2012爱思唯尔有限公司保留所有权利。
Undecaprenyl Pyrophosphate Synthase (UPPS) is a key enzyme that catalyzes the production of bactoprenols, which act as membrane anchors for the assembly of complex bacterial oligosaccharides. One of the major hurdles in understanding the assembly of oligosaccharide assembly is a lack of chemical tools to study this process, since bactoprenols and the resulting isoprenoid-linked oligosaccharides lack handles or chromophores for use in pathway analysis. Here we describe the isolation of a new UPPS from the symbiotic microorganism Bacteroides fragilis, a key species in the human microbiome. The protein was purified to homogeneity and utilized to accept a chromophore containing farnesyl diphosphate analogue as a substrate. The analogue was utilized by the enzyme and resulted in a bactoprenyl diphosphate product with an easy to monitor tag associated with it. Furthermore, the diphosphate is shown to be readily converted to monophosphate using a common molecular biology reagent. This monophosphate product allowed for the investigation of complex oligosaccharide biosynthesis, and was used to probe the activity of glycosyltransferases involved in the well characterized Campylobacter jejuni N-linked protein glycosylation. Novel reagents similar to this will provide key tools for the study of uncharacterized oligosaccharide assemblies, and open the possibility for the development of rapid screening methodology for these biosynthetic systems. (c) 2012 Elsevier Ltd. All rights reserved.