Structure-Function Analysis of UDP-Sugar: Polyisoprenyl Phosphate Sugar-1-Phosphate Transferases
Doctor of Philosophy
Microbiology and Immunology
Dr. Miguel Valvano
The synthesis of lipid-linked glycans is a conserved process in eukaryotes and prokaryotes that is initiated by two major enzyme families: the polyisoprenyl-phosphate hexose-1-phosphate transferases (PHPTs) and the polyisoprenyl-phosphate N-acetylaminosugar-1-phosphate transferases (PNPTs). These enzymes contain multiple membrane domains and transfer a sugar-1-phosphate from a nucleotide sugar precursor to a lipid carrier. The prototypic PNPT member used in this study is the E. coli WecA, which initiates the synthesis of O antigen and enterobacterial common antigen in Enterobacteriaceae by transferring N-acetylglucosamine-1-P to undecaprenyl phosphate (Und-P). We investigated the topology and function of the highly conserved VFMGD motif. Our results revealed that this motif faces the cytosol and defines a region in PNPTs that contributes to the active site, likely involved in the binding and/or recognition of the nucleotide moiety of the nucleoside phosphate precursor. The PHPT family member used in this study is the E. coli WcaJ, which transfers glucose-1-P to Und-P to initiate colanic acid synthesis. We provide the first detailed topological analysis of a PHPT member, which is inverted compared to the in silico topological predictions; the N-terminus, C-terminal tail and the large soluble loop all reside in the cytoplasm. We also found that the last membrane domain does not fully span the membrane and is likely ‘pinched in’. We further investigated the role of the N-terminal domain of WcaJ and our data suggest that it likely contributes to the protein folding and/or stability of PHPT family members. Together this work sheds light on the topological and mechanistic differences between these two major enzyme families and will guide further structural studies.
Furlong, Sarah E., "Structure-Function Analysis of UDP-Sugar: Polyisoprenyl Phosphate Sugar-1-Phosphate Transferases" (2013). Electronic Thesis and Dissertation Repository. 1629.