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Abstract
Capsular Polysaccharide A (CPSA) is a polymer of a tetrasaccharide unit found on the surface of the symbiotic gut bacteria Bacteroides fragilis. It has been suggested that CPSA is vital for maintaining a normal balance between the levels of Th1 and Th2 cells in a normal mammalian immune system. The gene locus responsible for CPSA biosynthesis has been identified. The operon has been proposed to encode one glycosyl-1-phosphate transferase (WcfS) and three glycosyltransferases (WcfN, -P and -Q), three sugar modifying enzymes (WcfM, WcfR and WcfO), a flippase (Wzx) and a polysaccharide polymerase (Wzy) based on bioinformatics tools. A route for the biosynthesis of CPSA is proposed. The initiating sugar transferase, WcfS has been previously identified and characterized. An in vitro method was used to enzymatically synthesize CPSA, which was assembled on a fluorescent analogue of the native bactoprenyl diphosphate anchor one sugar at a time. Utilizing reverse phase HPLC, functional characterization of the remaining glycosyltransferases, WcfN, WcfP and WcfQ involved in the CPSA biosynthesis was done. Also function of the hypothesized pyruvyltransferase WcfO was determined. This is the first study to characterize a pyruvyltransferase involved in polysaccharide biosynthesis from a prokaryote. The putative galactopyranose mutase WcfM was also characterized. The biosynthesis of the polysaccharide was also achieved in a single pot, compared to the multiple steps involved in chemical synthesis, displaying an enormous leap in the biosynthesis of complex molecules like CPSA.