Sharma Sunita, Erickson Katelyn M, Troutman Jerry M
Department of Chemistry, ‡The Center for Biomedical Engineering and Science, §Department of Biological Sciences, ∥Nanoscale Science Program, University of North Carolina at Charlotte , 9201 University City Blvd., Charlotte, North Carolina 28223, United States.
ACS Chem Biol. 2017 Jan 20;12(1):92-101. doi: 10.1021/acschembio.6b00931. Epub 2016 Nov 28.
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.
荚膜多糖A（CPSA）是一种在肠道共生菌脆弱拟杆菌表面发现的四糖重复单元聚合物，在自身免疫性疾病动物模型中具有治疗潜力。这种治疗潜力归因于其两性离子特性，该特性源自带正电荷的N-乙酰-4-氨基半乳糖（AADGal）和带负电荷的4,6-O-丙酮酰化半乳糖（PyrGal）。在本报告中，使用荧光聚异戊二烯化学探针实现了CPSA四糖重复单元的完整酶促组装。由CPSA生物合成基因簇编码的假定丙酮酰转移酶WcfO、吡喃半乳糖变位酶WcfM以及糖基转移酶WcfP和WcfN被异源表达并进行了功能表征。发现由WcfO催化的丙酮酸修饰发生在聚异戊二烯连接的二糖（AADGal-Gal）的半乳糖上，而不发生在与尿苷二磷酸（UDP）连接的半乳糖或一组硝基苯基半乳糖类似物上。还发现这种丙酮酸修饰是糖基转移酶WcfP将途径中的下一个糖N-乙酰半乳糖胺（GalNAc）掺入所必需的。半乳糖的丙酮酸缩醛修饰以前在多糖生物合成途径中尚未被探索，这项工作证明了这种修饰对重复单元组装的重要性。在产生聚异戊二烯连接的AADGal-PyrGal-GalNAc后，发现蛋白质WcfM和WcfN协同作用形成最终的四糖，其中WcfM形成UDP-呋喃半乳糖（Galf），WcfN将Galf转移到AADGal-PyrGal-GalNAc上。这项工作展示了在连续单锅反应中首次对CPSA四糖重复单元进行酶促组装。
