Feedback Inhibition of Bacterial Nucleotidyltransferases by Rare Nucleotide l-Sugars Restricts Substrate Promiscuity.

Bacterial glycomes are rich in prokaryote-specific or "rare" sugars that are absent in mammals. Like common sugars found across organisms, rare sugars are typically activated as nucleoside diphosphate sugars (NDP-sugars) by nucleotidyltransferases. In bacteria, the nucleotidyltransferase RmlA initiates the production of several rare NDP-sugars, which in turn regulate downstream glycan assembly through feedback inhibition of RmlA via binding to an allosteric site. In vitro, RmlA activates a range of common sugar-1-phosphates to produce NDP-sugars for biochemical and synthetic applications. However, our ability to probe bacterial glycan biosynthesis is hindered by limited chemoenzymatic access to rare NDP-sugars. We postulate that natural feedback mechanisms impact nucleotidyltransferase utility. Here, we use synthetic rare NDP-sugars to identify structural features required for regulation of RmlA from diverse bacterial species. We find that mutation of RmlA to eliminate allosteric binding of an abundant rare NDP-sugar facilitates the activation of noncanonical rare sugar-1-phosphate substrates, as products no longer affect turnover. In addition to promoting an understanding of nucleotidyltransferase regulation by metabolites, this work provides new routes to access rare sugar substrates for the study of important bacteria-specific glycan pathways.

Chemical Biology Tools for Modulating and Visualizing Gram-Negative Bacterial Surface Polysaccharides.

Bacterial cells present a wide diversity of saccharides that decorate the cell surface and help mediate interactions with the environment. Many Gram-negative cells express O-antigens, which are long sugar polymers that makeup the distal portion of lipopolysaccharide (LPS) that constitutes the surface of the outer membrane. This review highlights chemical biology tools that have been developed in recent years to facilitate the modulation of O-antigen synthesis and composition, as well as related bacterial polysaccharide pathways, and the detection of unique glycan sequences. Advances in the biochemistry and structural biology of O-antigen biosynthetic machinery are also described, which provide guidance for the design of novel chemical and biomolecular probes. Many of the tools noted here have not yet been utilized in biological systems and offer researchers the opportunity to investigate the complex sugar architecture of Gram-negative cells.