Enzymatic methods to phosphorylate bacteria-specific l-sugars and synthetic analogs for glycan engineering

Rhamnose is a 6-deoxy “rare” sugar that is abundant in plants and microbial natural products but absent in mammals. It is prevalent in various cell surface glycoconjugates, however little is known about its functions in mediating cell-cell interactions. In Gram- negative bacteria, L-Rha is a major component of many microbial cell surface polysaccharides which play a vital role in virulence and pathogenicity by mediating interactions between the bacterial cell and its environment. For metabolic incorporation into surface polysaccharides, L-Rha must be phosphorylated and activated as a nucleotide sugar for transfer by glycosyltransferases. In this study, we have expressed and purified a microbial sugar kinase and confirmed its activity as an L -sugar kinase. We designed and chemically synthesized probes of L-Rha containing bio- orthogonal functional groups, such as an azide and an alkyl diazirine, and performed an alanine scan on our sugar kinase to identify mutants capable of phosphorylating these synthesized analogs. As few routes towards L-sugar activation exist, this work provides the foundation for the enzymatic activation of L-sugar analogs, which may be used for applications both in vivo and in vitro. Overall, this work will expand our understanding of the specific roles that rare sugars play in host-pathogen interactions and provide new biocatalytic routes towards activated L-Rha and other “rare” sugars.