Mia Sheshova
New York University
Understanding the transfer of L-Rhamnose by bacterial glycosyltransferases
O-antigen (O-Ag), located in the outer leaflet of the outer membrane of Gram-negative bacteria, is a complex carbohydrate structure that is essential for bacterial virulence. Glysosyltransferases transfer the sugar unit onto a growing glycolipid chain and are important for understanding the assembly of O-Ag. Due to the obstacles associated with the purification of membrane-associated enzymes and the generation of glycolipid substrates, there are very few studies on the characterization of bacterial glycosyltransferases involved in rare sugar transfer in cell envelope assembly. We previously reported a gene of predicted similar function to the first glycosyltransferase in this pathway, Thermus thermophilus RfbF. In collaboration with Karen Allens’s lab from Boston University, we were able to obtain a X-ray crystal structure of RfbF in both the apo-form and co-complexed to dTDP, with a resolution of 2.5 and 2.7 Å, respectively. Guided by our structural studies, we are currently generating point mutants to investigate their impact on O-Ag expression in cells. Altogether, this work will lead to a better understanding behind the mechanism of the GT-A proteins for transferring rare sugars and will aid in the discovery of developing new strategies to stall the synthesis of bacterial virulence factors.
O-antigen (O-Ag), located in the outer leaflet of the outer membrane of Gram-negative bacteria, is a complex carbohydrate structure that is essential for bacterial virulence. Glysosyltransferases transfer the sugar unit onto a growing glycolipid chain and are important for understanding the assembly of O-Ag. Due to the obstacles associated with the purification of membrane-associated enzymes and the generation of glycolipid substrates, there are very few studies on the characterization of bacterial glycosyltransferases involved in rare sugar transfer in cell envelope assembly. We previously reported a gene of predicted similar function to the first glycosyltransferase in this pathway, Thermus thermophilus RfbF. In collaboration with Karen Allens’s lab from Boston University, we were able to obtain a X-ray crystal structure of RfbF in both the apo-form and co-complexed to dTDP, with a resolution of 2.5 and 2.7 Å, respectively. Guided by our structural studies, we are currently generating point mutants to investigate their impact on O-Ag expression in cells. Altogether, this work will lead to a better understanding behind the mechanism of the GT-A proteins for transferring rare sugars and will aid in the discovery of developing new strategies to stall the synthesis of bacterial virulence factors.