2008 OSU Molecular Life Sciences
Interdisciplinary Graduate Programs Symposium

 

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Poster number 115 submitted by Wen Yi

Oligosaccharide repeating unit substrates for probing bacterial polysaccharide biosynthesis

Wen Yi (Biochemistry, OSU), Robert Woodward (Chemistry, OSU), Peng G. Wang (Biochemistry, OSU)

Abstract:
Bacterial cell surface polysaccharides mediate interactions between the bacterial cells and their environment, facilitating the colonization of the cells to the host and serve as molecular mimicry to evade the host immune attack. Thus understanding how the polysaccharides are synthesized is a fundamentally important biological question with significant medical implications.
In spite of the remarkably structural diversity of polysaccharides, only a handful of biosynthetic mechanisms are known, among which the “wzy-dependent” mechanism is the most widely used based on previous genetic studies. In this mechanism, oligosaccharide repeating unit substrates (oligosaccharides linked to undecaprenyl diphosphate) are firstly synthesized at the cytoplasm by specific glycosyltranferases, and subsequently polymerized by polymerase Wzy in the periplasm. Currently there is no direct biochemical information on the mechanism of the polymerization process. The effort to biochemically characterize polymerization is hampered by the lack of repeating unit substrates. In this study, we used E. coli O86 as a mode system and prepared a series of repeating unit substrate analogs using chemo-enzymatic synthesis. We identified pofour glycosyltransferases in the E. coli O86 polysaccharide synthetic gene cluster that are involved in the biosynthesis of repeating unit substrates. GalNAc-PP-Lipid synthetic precursors were then chemically synthesized. Three different lipid structures were constructed: saturated 11-carbon lipid, 15-carbon farnesyl lipid and 35-carbon heptaprenyl lipid. With GalNAc-PP-lipid precursors as substrates, in vitro sequential enzymatic glycosylation was carried out with four glycosyltransferases to synthesize the entire pentasaccharide repeating unit structure. The structures of each repeating unit analogs were confirmed by NMR and MS. This study provides the direct biochemical evidence for the repeating unit biosynthesis and lays the groundwork for probing the polymerization mechanism.

Keywords: polysaccharide, polymerization, glycosyltransferase