Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A.
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ABSTRACT: 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.
SUBMITTER: Sharma S
PROVIDER: S-EPMC5292236 | biostudies-literature | 2017 Jan
REPOSITORIES: biostudies-literature
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