Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A.
Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A.
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DOI：
10.1021/acschembio.6b00931
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发表时间：
2017-01-20
影响因子：
4
通讯作者：
Troutman JM
中科院分区：
文献类型：
--
作者：
Sharma S;Erickson KM;Troutman JM
荚膜多糖A（CPSA）是一种在肠道共生体脆弱拟杆菌表面发现的四糖重复单元聚合物，在自身免疫性疾病的动物模型中具有治疗潜力。这种治疗潜力已被归功于其源自带正电荷的N-乙酰基-4-氨基半乳糖胺（AADGal）和带负电荷的4，6-O-乙酰基化半乳糖（PyrGal）的两性离子特性。在这份报告中，使用荧光聚异戊二烯化学探针，实现了CPSA四糖重复单元的完全酶促组装。提出的乙酰基转移酶，WcfO，吡喃半乳糖苷，WcfM，和糖基转移酶，WcfP和WcfN，编码的CPSA生物合成基因簇异源表达和功能特征。丙酮酸修饰，催化WcfO，被发现发生在半乳糖的聚类异戊二烯连接的二糖（AADGal-Gal），并没有发生在半乳糖连接的尿苷二磷酸（UDP）或一组硝基苯-半乳糖类似物。还发现这种丙酮酸修饰是通过糖基转移酶WcfP掺入途径N-乙酰半乳糖胺（GalNAc）中的下一个糖所需的。半乳糖的丙酮酸缩醛修饰以前没有在多糖生物合成途径的背景下探索过，这项工作证明了这种修饰对重复单元组装的重要性。在产生聚类异戊二烯连接的AADGal-PyrGal-GalNAc后，发现蛋白质WcfM和WcfN协同作用以形成最终的四糖，其中WcfM形成UDP-呋喃半乳糖（Galf），WcfN将Galf转移至AADGal-PyrGal-GalNAc。这项工作证明了CPSA四糖重复单元在连续单锅反应中的第一次酶促组装。
Capsular polysaccharide A (CPSA) is a four-sugar repeating unit polymer found on the surface of the gut symbiont Bacteroides fragilis that has therapeutic potential in animal models of autoimmune disorders. This therapeutic potential has been credited to its zwitterionic character derived from a positively charged N-acetyl-4-aminogalactosamine (AADGal) and a negatively charged 4,6-O-pyruvylated galactose (PyrGal). In this report, using a fluorescent polyisoprenoid chemical probe, the complete enzymatic assembly of the CPSA tetrasaccharide repeat unit is achieved. The proposed pyruvyltransferase, WcfO, galactopyranose mutase, WcfM, and glycosyltransferases, WcfP and WcfN, encoded by the CPSA biosynthesis gene cluster were heterologously expressed and functionally characterized. Pyruvate modification, catalyzed by WcfO, was found to occur on galactose of the polyisoprenoid-linked disaccharide (AADGal-Gal), and did not occur on galactose linked to uridine diphosphate (UDP) or a set of nitrophenyl-galactose analogues. This pyruvate modification was also found to be required for the incorporation of the next sugar in the pathway N-acetylgalactosamine (GalNAc) by the glycosyltransferase WcfP. The pyruvate acetal modification of a galactose has not been previously explored in the context of a polysaccharide biosynthesis pathway, and this work demonstrates the importance of this modification to repeat unit assembly. Upon production of the polyisoprenoid-linked AADGal-PyrGal-GalNAc, the proteins WcfM and WcfN were found to work in concert to form the final tetrasaccharide, where WcfM formed UDP-galactofuranose (Galf) and WcfN transfers Galf to the AADGal-PyrGal-GalNAc. This work demonstrates the first enzymatic assembly of the tetrasaccharide repeat unit of CPSA in a sequential single pot reaction.